Ammonium transporter promoter for gene expression in oleaginous yeast

ABSTRACT

The promoter region associated with the  Yarrowia lipolytica  ammonium transporter (yat1) gene has been found to be particularly effective for the expression of heterologous genes in oleaginous yeast. The promoter regions of the instant invention have been shown to be advantageously inducible under oleaginous conditions (i.e., nitrogen limitation) and are useful to drive expression of genes involved in the production of ω-3 and ω-6 fatty acids.

FIELD OF THE INVENTION

This invention is in the field of biotechnology. More specifically, this invention pertains to a promoter region isolated from Yarrowia lipolytica that is useful for gene expression in oleaginous yeast.

BACKGROUND OF THE INVENTION

Oleaginous yeast are defined as those organisms that are naturally capable of oil synthesis and accumulation, wherein oil accumulation ranges from at least about 25% up to about 80% of the cellular dry weight. Genera typically identified as oleaginous yeast include, but are not limited to: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. More specifically, illustrative oil-synthesizing yeast include: Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus, R. graminis and Yarrowia lipolytica (formerly classified as Candida lipolytica).

The technology for growing oleaginous yeast with high oil content is well developed (for example, see EP 0 005 277B1; Ratledge, C., Prog. Ind. Microbiol. 16:119-206 (1982)). And, these organisms have been commercially used for a variety of purposes in the past. For example, various strains of Yarrowa lipolytica have historically been used for the manufacture and production of: isocitrate lyase; lipases; polyhydroxy-alkanoates; citric acid; erythritol; 2-oxoglutaric acid; γ-decalactone; γ-dodecalactone; and pyruvic acid. More recently, however, the natural abilities of oleaginous yeast have been enhanced by advances in genetic engineering, resulting in organisms capable of producing polyunsaturated fatty acids (“PUFAs”). Specifically, Picataggio et al. have demonstrated that Y. lipolytica can be engineered for production of ω-3 and ω-6 fatty acids, by introducing and expressing genes encoding the ω-3/ω-6 biosynthetic pathway (see WO 2004/101757 and co-pending U.S. patent application Ser. No. 60/624,812).

Recombinant production of any heterologous protein is generally accomplished by constructing an expression cassette in which the DNA coding for the protein of interest is placed under the control of appropriate regulatory sequences (i.e., promoters) suitable for the host cell. The expression cassette is then introduced into the host cell (usually by plasmid-mediated transformation or targeted integration into the host genome) and production of the heterologous protein is achieved by culturing the transformed host cell under conditions necessary for the proper function of the promoter contained within the expression cassette. Thus, the development of new host cells (e.g., oleaginous yeast) for recombinant production of proteins generally requires the availability of promoters that are suitable for controlling the expression of a protein of interest in the host cell.

A variety of strong promoters have been isolated from Yarrowia lipolytica that are useful for heterologous gene expression in yeast. For example, U.S. Pat. No. 4,937,189 and EP220864 (Davidow et al.) disclose the sequence of the XPR2 gene (which encodes an inducible alkaline extracellular protease) and upstream promoter region for use in expression of heterologous proteins. U.S. Pat. No. 6,265,185 (Muller et al.) describes promoters for the translation elongation factor EF1-α (TEF) protein and ribosomal protein S7 that are suitable for expression cloning in yeast and heterologous expression of proteins. These promoters were improved relative to the XPR2 promoter, when tested for yeast promoter activity on growth plates (Example 9, U.S. Pat. No. 6,265,185) and based on their activity in the pH range of 4-11. WO 2005/003310 describe regulatory sequences (e.g., promoters, introns) of the glyceraldehyde-3-phosphate dehydrogenase (gpd) and phosphoglycerate mutase (gpm) genes; WO 2005/049805 describes regulatory sequences (e.g., promoters, introns) of the fructose-bisphosphate aldolase (fba) gene; and, co-pending U.S. patent application Ser. No. 60/610,060 describes promoters of the glycerol-3-phosphate O-acyltransferase (gpat) gene. Similarly, Juretzek et al. (Biotech. Bioprocess Eng., 5:320-326 (2000)) compares the glycerol-3-phosphate dehydrogenase (G3P), isocitrate lyase (ICL1), 3-oxo-acyl-CoA thiolase (POT1) and acyl-CoA oxidase (POX1, POX2 and POX5) promoters with respect to their regulation and activities during growth on different carbon sources.

Despite the utility of these known promoters, however, there is a need for new improved yeast promoters for metabolic engineering of yeast (oleaginous and non-oleaginous) and for controlling the expression of heterologous genes in yeast. Furthermore, possession of a suite of promoters that are regulatable under a variety of natural growth and induction conditions in yeast will play an important role in industrial settings, wherein it is desirable to express heterologous polypeptides in commercial quantities in said hosts for economical production of those polypeptides. Thus, it is an object of the present invention to provide such promoters that will be useful for gene expression in a variety of yeast cultures, and preferably in Yarrowia sp. cultures and other oleaginous yeast.

Applicants have solved the stated problem by identifying the YAT1 promoter from Yarrowia lipolytica, which is responsible for driving expression of the gene (yat1) encoding an ammonium transporter (YAT1). Advantageously, the promoter is useful for regulated expression of heterologous genes in Yarrowia, has improved activity with respect to the TEF promoter and is inducible under oleaginous conditions (i.e., nitrogen limitation).

SUMMARY OF THE INVENTION

The present invention provides methods for the regulated expression of a coding region of interest in a transformed yeast, using a promoter region of an ammonium transporter (yat1) gene. Accordingly the invention provides a method for the expression of a coding region of interest in a transformed yeast comprising:

-   -   a) providing a transformed yeast having a genetic construct         comprising:         -   (i) a promoter region of a Yarrowia yat1 gene; and,         -   (ii) a coding region of interest expressible in the yeast             cell;     -   wherein the promoter region is operably linked to the coding         region of interest; and,     -   b) growing the transformed yeast of step (a) under conditions         whereby the genetic construct of step (a) is expressed.

In another embodiment mutant yat1 promoters are provided exhibiting mutations that demonstrate between 20% and 400% increased promoter activity as compared with the wildtype promoter.

In an alternate embodiment the invention provides a method for the production of an ω-3 or an ω-6 fatty acid comprising:

-   -   a) providing a transformed oleaginous yeast comprising a genetic         construct, comprising:         -   (i) a promoter region of a Yarrowia yat1 gene; and,         -   (ii) a coding region encoding at least one enzyme of the             ω-3/ω-6 fatty acid biosynthetic pathway;     -   wherein the promoter region and coding region are operably         linked;     -   b) culturing the transformed oleaginous yeast of step (a) under         conditions whereby the at least one enzyme of the ω-3/ω-6 fatty         acid biosynthetic pathway is expressed and a ω-3 or ω-6 fatty         acid is produced; and,     -   c) optionally recovering the ω-3 or ω-6 fatty acid.

Preferred conditions suitable to induce expression of the genetic constructs of the invention are conditions of nitrogen-limitation; and, in a more preferred embodiment, the nitrogen-limiting conditions are those that induce oleaginy.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

FIG. 1 provides plasmid maps for the following: (A) pY5-30; (B) pDMW214; (C) pYGPAT-GUS; and (D) pYAT-GUS, respectively.

FIG. 2 graphically represents the relationship between SEQ ID NOs:12, 15, 22, 23 and 24, each of which relates to glycerol-3-phosphate O-acyltransferase (GPAT) in Y. lipolytica.

FIG. 3A diagrams the development of Yarrowia lipolytica strain Y2034, producing up to 10% ARA in the total lipid fraction. FIG. 3B provides a plasmid map for pKUNF12T6E; and FIG. 3C provides a plasmid map for pDMW232.

FIGS. 4A, 4B and 4C illustrate the relative promoter activities of YAT1, TEF, GPAT and FBAIN in Y. lipolytica grown in various media as determined by histochemical staining.

FIG. 5 illustrates the ω-3/ω-6 fatty acid biosynthetic pathway.

The invention can be more fully understood from the following detailed description and the accompanying sequence descriptions, which form a part of this application.

The following sequences comply with 37 C.F.R. §1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NOs:1-3, 10-12, 15, 23, 24, and 27-37 correspond to ORFs (i.e., encoding genes or proteins), promoters and plasmids, as identified in Table 1. TABLE 1 Summary Of Nucleotide And Protein SEQ ID Numbers Protein Nucleotide SEQ ID Description SEQ ID NO NO Yarrowia lipolytica ammonium transporter  1  2 (yat1) (1461 bp) (486 AA) (GenBank Accession No. XM_504457) YAT1 promoter  3 —  (778 bp) Plasmid pY5-30 10 — (8953 bp) FBAIN promoter 11 —  (973 bp) Yarrowia lipolytica ORF YALI-CDS1055.1 — 12 (GenBank Accession No. CAG81570) (727 AA) Yarrowia lipolytica glycerol-3-phosphate O- 15 — acyltransferase (gpat) gene (2184 bp) Yarrowia lipolytica gpat gene: −1678 to 23 — +2181 region (3862 bp) GPAT promoter 24 — (1130 bp) Plasmid pKUNF12T6E 27 — 12,649 bp)  Synthetic elongase gene derived from 28 29 Mortierella alpina, codon-optimized for  (957 bp) (318 AA) expression in Yarrowia lipolytica Synthetic Δ6 desaturase, derived from 30 31 Mortierella alpina, codon-optimized for (1374 bp) (457 AA) expression in Yarrowia lipolytica FBA promoter 32 — (1001 bp) Fusarium moniliforme Δ12 desaturase 33 34 (1434 bp) (477 AA) Synthetic elongase gene derived from 35 36 Thraustochytrium aureum, codon-optimized  (819 bp) (272 AA) for expression in Yarrowia lipolytica Plasmid pDMW232 37 — (10,945 bp)  

SEQ ID NOs:4, 5 and 6 correspond to BD-Clontech's Creator Smart® cDNA library kit primers SMART IV oligonucleotide, CDSIII/3′ PCR primer and 5′-PCR primer, respectively.

SEQ ID NO:7 corresponds to the M13 forward primer used for sequencing of Y. lipolytica cDNA.

SEQ ID NOs:8 and 9 correspond to primers 27203-F and 27203-R, respectively, used for amplifying the 5′ upstream region of the yat1 gene.

SEQ ID NOs:13 and 14 correspond to the degenerate primers YGPAT-F and YGPAT-R, respectively, used for amplifying Y. lipolytica ORF YALI-CDS1055.1.

SEQ ID NOs:16 and 17 correspond to the Genome Walker adaptor used to isolate the GPAT promoter region by genome-walking.

SEQ ID NOs:18-21 correspond to the PCR primers used in genome-walking: Adaptor-1, YGPAT-5R-1, Nested Adaptor Primer 2 and YGPAT-5R-2, respectively.

SEQ ID NO:22 corresponds to a 1781 bp fragment contained within plasmid pEcoRV-G-5, the fragment containing a 1678 bp region upstream of the translation initiation codon ‘ATG’ of the gpat gene.

SEQ ID NOs:25 and 26 correspond to primers GPAT-5-1 and GPAT-5-2, respectively, used to amplify the GPAT promoter.

DETAILED DESCRIPTION OF THE INVENTION

In support of the invention described herein the following commonly owned patents, and patent applications are incorporated herein by reference in their entirety: 10/840,579 (filed May 6, 2004), 10/869,630 (filed Jun. 16, 2004), 10/987,548 (filed Nov. 12, 2004), U.S. Patent Provisional Application No. 60/610,060 (filed Sep. 15, 2004), and U.S. Patent Provisional Application No. 60/624,812 (filed Nov. 4, 2004).

In accordance with the subject invention, Applicants describe the characterization of a promoter from an oleaginous yeast, Yarrowia lipolytica. This promoter region, isolated upstream of the ammonium transporter (yat1) gene and inducible under oleaginous conditions (i.e., nitrogen limitation), is useful for genetic engineering in Y. lipolytica and other yeast for the production of heterologous polypeptides.

Preferred heterologous polypeptides of the present invention are those that are involved in the synthesis of microbial oils and particularly polyunsaturated fatty acids (PUFAs). PUFAs, or derivatives thereof, made by the methodology disclosed herein can be used in many applications. For example, the PUFAs can be used as dietary substitutes, or supplements, particularly infant formulas, for patients undergoing intravenous feeding or for preventing or treating malnutrition. Alternatively, the purified PUFAs (or derivatives thereof) may be incorporated into cooking oils, fats or margarines formulated so that in normal use the recipient would receive the desired amount for dietary supplementation. The PUFAs may also be incorporated into infant formulas, nutritional supplements or other food products, and may find use as anti-inflammatory or cholesterol lowering agents. Optionally, the compositions may be used for pharmaceutical use (human or veterinary). In this case, the PUFAs are generally administered orally but can be administered by any route by which they may be successfully absorbed, e.g., parenterally (e.g., subcutaneously, intramuscularly or intravenously), rectally, vaginally or topically (e.g., as a skin ointment or lotion).

Thus, the present invention advances the art by providing methods for the regulated expression of a coding region of interest in a transformed yeast comprising: a) providing a transformed yeast having a chimeric gene comprising (i) a promoter region of a Yarrowia yat1 gene; and, (ii) a coding region of interest expressible in the yeast, wherein the promoter region is operably linked to the coding region of interest; b) growing the transformed yeast of step (a) under conditions whereby the chimeric gene is expressed; and, c) optionally isolating the gene product from the cultivation medium. In preferred embodiments, the YAT1 promoter region comprises a sequence as set forth in SEQ ID NO:3.

Definitions

In this disclosure, a number of terms and abbreviations are used. The following definitions are provided.

“Ammonium transporter” is abbreviated YAT.

“Open reading frame” is abbreviated ORF.

“Polymerase chain reaction” is abbreviated PCR.

“Polyunsaturated fatty acid(s)” is abbreviated PUFA(s).

The term “oleaginous” refers to those organisms that tend to store their energy source in the form of lipid (Weete, In: Fungal Lipid Biochemistry, 2^(nd) Ed., Plenum, 1980). Generally, the cellular PUFA content of these microorganisms follows a sigmoid curve, wherein the concentration of lipid increases until it reaches a maximum at the late logarithmic or early stationary growth phase and then gradually decreases during the late stationary and death phases (Yongmanitchai and Ward, Appl. Environ. Microbiol. 57:419-25 (1991)).

The term “oleaginous yeast” refers to those microorganisms classified as yeast that can accumulate at least 25% of their dry cell weight as oil. Examples of oleaginous yeast include (but are no means limited to) the following genera: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.

The term “ammonium transporter” refers to a family of transporters whose physiological role within a cell is to scavenge external ammonium for use as a nitrogen source (and, under some circumstances, to incorporate ammonium that leaks out of cells), based on studies of the MEP family of ammonium transporters in Saccharomyces cerevisiae (A. M. Marini et al., EMBO J., 13(15):3456-3463 (1994); Mol Cell Biol 17(8):4282-93 (1997)). In general, the proteins are subject to nitrogen control and each has different kinetic properties, specificities and regulation; for example, the three S. cerevisiae isozymes are characterized as follows: MEP1, a low affinity/high capacity ammonia transporter (K_(m), 5-10 μM); MEP2, a high affinity/low capacity ammonia transporter (K_(m), 1-2 μM); and MEP3, a low affinity ammonia transporter (K_(m), 1.4-2.1 mM). Other known members of this family include: the high affinity ammonium transporter (amt1), the ammonium and methylammonium transport system, the putative ammonium transporter amtB and nrgA.

As used herein, the term “YAT1” refers to an ammonium transporter enzyme (TC 2.A.49) encoded by the yat1 gene and isolated from Yarrowia lipolytica (GenBank Accession No. XM_(—)504457). The sequence disclosed therein (/locus_tag=“YALI0E27203g”) was identified as a hypothetical protein having similarity “to sp|P41948 Saccharomyces cerevisiae YNL142w MEP2, hypothetical start”. The Y. lipolytica yat1 gene is presented herein as SEQ ID NO:1, while the corresponding YAT1 protein is provided herein as SEQ ID NO:2.

The term “YAT1 promoter” or “YAT1 promoter region” refers to the 5′ upstream untranslated region in front of the ‘ATG’ translation initiation codon of yat1 and that is necessary for expression. An example of a suitable YAT1 promoter region is provided as SEQ ID NO:3, but this is not intended to be limiting in nature. One skilled in the art will recognize that since the exact boundaries of the YAT1 promoter sequence have not been completely defined, DNA fragments of increased or diminished length may have identical promoter activity.

The term “GPAT” refers to a glycerol-3-phosphate O-acyltransferase enzyme (E.C. 2.3.1.15) encoded by the gpat gene and which converts acyl-CoA and sn-glycerol 3-phosphate to CoA and 1-acyl-sn-glycerol 3-phosphate (the first step of phospholipid biosynthesis). The term “GPAT promoter” or “GPAT promoter region” refers to the 5′ upstream untranslated region in front of the ‘ATG’ translation initiation codon of gpat and that is necessary for expression. One example of a suitable GPAT promoter region is provided as SEQ ID NO:24, but this is not intended to be limiting in nature (see co-pending U.S. patent application Ser. No. 60/610,060).

The term “FBA1” refers to a fructose-bisphosphate aldolase enzyme (E.C. 4.1.2.13) encoded by the fba1 gene and which converts D-fructose 1,6-bisphosphate into glycerone phosphate and D-glyceraldehyde 3-phosphate. The term “FBAIN promoter” or “FBAIN promoter region” refers to the 5′ upstream untranslated region in front of the ‘ATG’ translation initiation codon of fba1 and that is necessary for expression, plus a portion of 5′ coding region comprising an intron of the fba1 gene. An example of a suitable FBAIN promoter region is provided as SEQ ID NO:11, but this is not intended to be limiting in nature (see WO 2005/049805).

The term “promoter activity” will refer to an assessment of the transcriptional efficiency of a promoter. This may, for instance, be determined directly by measurement of the amount of mRNA transcription from the promoter (e.g., by Northern blotting or primer extension methods) or indirectly by measuring the amount of gene product expressed from the promoter, as foe example by histochemical means.

The term “conditions of nitrogen-limitation” refers to a medium having a low concentration of nitrogen, wherein the nitrogen may be supplied from an inorganic (e.g., (NH₄)₂SO₄) or organic (e.g., urea or glutamate) source, or a medium having no nitrogen source. Although one skilled in the art will be able to determine an appropriate low concentration of nitrogen suitable to induce the YAT1 promoter of the present invention, in one embodiment a preferred medium would be one characterized as having a high carbon to nitrogen (i.e., C:N) ratio and about 0.1% or less ammonium sulfate or other suitable ammonium salts.

As used herein, an “isolated nucleic acid molecule” is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid molecule in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

The term “genetic construct” is a non-limiting term meaning any contiguous series of nucleic acids capable of being expressed in a host organism. A genetic construct may include but is not limited to an open reading frame, an open reading frame operably linked to regulatory sequences, or a wildtype or mutant gene. Genetic constructs may encode poylpeptides or be nucleic acid fragments or molecules that are oriented for antisense expression.

A nucleic acid molecule is “hybridizable” to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA molecule, when a single-stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments (such as homologous sequences from distantly related organisms), to highly similar fragments (such as genes that duplicate functional enzymes from closely related organisms). Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65 ° C. An additional set of stringent conditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.

Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least about 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.

A “substantial portion” of an amino acid or nucleotide sequence is that portion comprising enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1993)). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to identify putatively a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene-specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid molecule comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid molecule comprising the sequence.

The instant specification teaches nucleotide sequences encoding a particular microbial promoter region. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.

The term “oligonucleotide” refers to a nucleic acid, generally of at least 18 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule. In one embodiment, a labeled oligonucleotide can be used as a “probe” to detect the presence of a nucleic acid according to the invention. Thus, the term “probe” refers to a single-stranded nucleic acid molecule that can base pair with a complementary single-stranded target nucleic acid to form a double-stranded molecule. The term “label” will refer to any conventional molecule which can be readily attached to mRNA or DNA and which can produce a detectable signal, the intensity of which indicates the relative amount of hybridization of the labeled probe to the DNA fragment.

The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the instant invention also includes isolated nucleic acid molecules that are complementary to the complete sequences as reported in the accompanying Sequence Listing, as well as those substantially similar nucleic acid sequences.

The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in: 1.) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences is performed using the Clustal method of alignment (Higgins and Sharp, CABIOS. 5:151-153 (1989)) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method are: KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

Suitable promoter regions (isolated polynucleotides of the present invention) encode promoter regions that are at least about 70% identical, preferably at least about 75% identical, and more preferably at least about 80% identical to the nucleotide sequences reported herein. Preferred nucleic acid molecules are about 85% identical to the nucleotide sequences reported herein, more preferred nucleic acid molecules are at least about 90% identical, and most preferred are nucleic acid molecules at least about 95% identical to the nucleotide sequences reported herein. Suitable promoter regions not only have the above homologies but typically are at least 50 nucleotides in length, more preferably at least 100 nucleotides in length, more preferably at least 250 nucleotides in length, and more preferably at least 500 nucleotides in length.

“Codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

“Chemically synthesized”, as related to a sequence of DNA, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well-established procedures; or, automated chemical synthesis can be performed using one of a number of commercially available machines. “Synthetic genes” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form gene segments that are then enzymatically assembled to construct the entire gene. Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell, where sequence information is available.

“Gene” refers to a nucleic acid molecule that expresses a specific protein, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. Chimeric genes of the present invention will typically comprise a YAT1 promoter region operably linked to a coding region of interest. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure. A “codon-optimized gene” is a gene having its frequency of codon usage designed to mimic the frequency of preferred codon usage of the host cell.

“Coding sequence” refers to a DNA sequence that codes for a specific amino acid sequence.

“Suitable regulatory sequences” refer to transcriptional and translational nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures.

“Promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

The term “mutant promoter” is defined herein as a promoter having a nucleotide sequence comprising a substitution, deletion, and/or insertion of one or more nucleotides relative to the parent promoter, wherein the mutant promoter has more or less promoter activity than the corresponding parent promoter. The term “mutant promoter” will encompass natural variants and in vitro generated variants obtained using methods well known in the art (e.g., classical mutagenesis, site-directed mutagenesis and “DNA shuffling”).

The term “3′non-coding sequences” or “transcription terminator” refers to DNA sequences located downstream of a coding sequence. This includes polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The 3′ region can influence the transcription, RNA processing or stability, or translation of the associated coding sequence.

“RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as the mature RNA. “Messenger RNA” or “mRNA” refers to the RNA that is without introns and that can be translated into protein by the cell. “cDNA” refers to a double-stranded DNA that is complementary to, and derived from, mRNA. “Sense” RNA refers to RNA transcript that includes the mRNA and so can be translated into protein by the cell. “Antisense RNA” refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065; WO 99/28508). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, or the coding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNA that is not translated and yet has an effect on cellular processes.

The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid molecule so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a coding sequence. Expression may also refer to translation of mRNA into a polypeptide.

“Transformation” refers to the transfer of a nucleic acid molecule into a host organism, resulting in genetically stable inheritance. The nucleic acid molecule may be a plasmid that replicates autonomously, for example; or, it may integrate into the genome of the host organism. Host organisms containing the transformed nucleic acid molecules are referred to as “transgenic” or “recombinant” or “transformed” organisms.

The terms “plasmid”, “vector” and “cassette” refer to an extra chromosomal element often carrying genes that are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA fragments. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell. “Expression cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.

The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. “Sequence analysis software” may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: 1.) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); and 4.) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Suhai, Sandor, Ed. Plenum: New York, N.Y.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified. As used herein “default values” will mean any set of values or parameters that originally load with the software when first initialized.

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987).

Identification of YAT1 and the YAT1 Promoter in Yarrowia lipolytica

Ammonium transporters are a family of transporters whose physiological role within a cell is to scavenge external ammonium for use as a nitrogen source (and, under some circumstances, to incorporate ammonium that leaks out of cells). In general, the proteins are subject to nitrogen control and each have different kinetic properties, specificities and regulation; for example, the three S. cerevisiae MEP1, MEP2, and MEP3 isozymes have been extensively characterized by A. M. Marini et al. (EMBO J., 13(15): 3456-3463 (1994); Mol Cell Biol 17(8):4282-93 (1997)) as a low affinity/high capacity ammonia transporter, a high affinity/low capacity ammonia transporter, and a low affinity ammonia transporter, respectively.

A cDNA library was constructed from Yarrowia lipolytica cells grown in an oleaginous medium containing no nitrogen source for 4 hrs (i.e., oleaginous conditions that promoter oil production). The relative abundance of each species of cDNA was then examined to identify those genes that were highly expressed under the oleaginous conditions. One of the genes that appeared multiple times (16/9984 or 0.16%) was GenBank Accession No. XM_(—)504457, locus_tag=“YALI0E27203g”. This gene was annotated as a hypothetical protein having similarity “to sp|P41948 Saccharomyces cerevisiae YNL142w MEP2 [the high affinity, low capacity ammonia transporter; GenBank Accession No. X83608], hypothetical start”.

Based on sequence comparison to the MEP1, MEP2 and MEP3 genes (supra), the Applicants hypothesized that the Y. lipolytica ORF likely encoded an ammonium transporter and designated the gene as the “yat1” gene (SEQ ID NOs:1 and 2). The “YAT1 promoter” was identified as the 5′ upstream untranslated region in front of the ‘ATG’ translation initiation codon of yat1 and that is necessary for expression. According to the invention here, this putative YAT1 promoter region will be useful for driving expression of any suitable coding region of interest in a transformed yeast cell. In general, a promoter useful in an oleaginous yeast should meet the following criteria:

-   -   1.) Strength. A strong yeast promoter is a necessary premise for         a high expression level, and the low copy number of the ars18         (Fournier, P. et al., Yeast 7:25-36 (1991)) based expression         vectors or chimeric genes integrated into the genome makes this         demand even more important when Y. lipolytica is used as the         host organism.     -   2.) Activity in a medium suitable for expression of the coding         region of interest, and high enzymatic activity of that coding         region of interest.     -   3.) pH Tolerance. If the coding region of interest is known to         be produced only in e.g., an acidic environment, then the         promoter operably linked to said coding region of interest must         function at the appropriate pH. pH tolerance is of course         limited by the tolerance of the host organism.     -   4.) Inducibility. A tightly regulated yeast promoter makes it         possible to separate the growth stage from the expression stage,         thereby enabling expression of products that are known to         inhibit cell growth.     -   5.) Activity in the stationary phase of growth in oleaginous         yeast hosts for accumulation of PUFAs.

Additionally, it is preferable for novel yeast promoters to possess differences in activity with respect to the known Yarrowia lipolytica TEF (U.S. Pat. No. 6,265,185), XPR2 (U.S. Pat. No. 4,937,189; EP220864; EP832258), GPD, GPM (U.S. Ser. No. 10/869,630), FBA, FBAIN (U.S. Ser. No. 10/987,548), and GPAT (U.S. Provisional Patent Application No. 60/610,060) promoters and/or the G3P, ICL1, POT1, POX1, POX2 and POX5 promoters (Juretzek et al., Biotech. Bioprocess Eng., 5:320-326 (2000)).

A comparative study of the TEF, FBAIN and GPAT promoters and the YAT1 promoter of the instant invention is provided in Examples 8 and 9. As shown in Table 7 of Example 9, the yeast promoter of the present invention has improved activity compared to the TEF promoter (i.e., 1.3:1 nmoles of 4-methylumbelliferone per minute per mg of total protein) under conditions where nitrogen is not limiting (i.e., minimal medium containing ammonium sulfate as the nitrogen source). Furthermore, under conditions of nitrogen limitation, the YAT1 promoter is significantly induced such that the activity is ˜20-28 times greater than that of TEF; thus, when grown in such medium, the activity of the YAT1 promoter is comparable to that of the strong FBAIN promoter. Based on these results, the Applicants characterize the YAT1 promoter as the first promoter identified within Yarrowia that is inducible under oleaginous conditions (i.e., nitrogen limitation).

An example of a suitable YAT1 promoter region is provided as SEQ ID NO:3 (comprising the −778 to −1 region of the Y. lipolytica yat1 gene (wherein the ‘A’ position of the ‘ATG’ translation initiation codon is designated as +1)), but this is not intended to be limiting in nature. One skilled in the art will recognize that since the exact boundaries of the YAT1 promoter sequence have not been completely defined, DNA fragments of increased or diminished length may have identical promoter activity. For example, in an alternate embodiment, the YAT1 promoter will comprise nucleotides −500 to −1 of SEQ ID NO:3, thereby permitting relatively strong promoter activity; in another embodiment, the −100 to −1 region of SEQ ID NO:3 should be sufficient for basal activity of the promoter. Likewise, the promoter region of the invention may comprise additional nucleotides to those specified above. For example, the promoter sequences of the invention may be constructed on the basis of the −1000 to +1 region of the yat1 gene (e.g., nucleotide bases 3,222,879 to 3,223,879 of GenBank Accession No. CR382131, comprising the complete nucleotide sequence of chromosome E of strain CLIB99 of Y. lipolytica).

In alternate embodiments mutant promoters may be constructed, wherein the DNA sequence of the promoter has one or more nucleotide substitutions (i.e., deletions, insertions, substitutions, or addition of one or more nucleotides in the sequence) which do not effect (in particular impair) the yeast promoter activity. Regions that can be modified without significantly affecting the yeast promoter activity can be identified by deletion studies. A mutant promoter of the present invention has at least about 20%, preferably at least about 40%, more preferably at least about 60%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 100%, more preferably at least about 200%, more preferably at least about 300% and most preferably at least about 400% of the promoter activity of the YAT1 promoter region described herein as SEQ ID NO:3.

Methods for mutagenesis are well known in the art and suitable for the generation of mutant promoters. For example, in vitro mutagenesis and selection, PCR based random mutagenesis, site-directed mutagenesis or other means can be employed to obtain mutations of the naturally occurring promoter of the instant invention (wherein such mutations may include deletions, insertions and point mutations, or combinations thereof). This would permit production of a putative promoter having a more desirable level of promoter activity in the host cell. Or, if desired, the regions of a nucleotide of interest important for promoter activity can be determined through routine mutagenesis, expression of the resulting mutant promoters and determination of their activities. An overview of these techniques is described in U.S. Ser. No. 10/869630. All such mutant promoters that are derived from the instant YAT1 promoter described herein are within the scope of the present invention.

Promoter activity is typically measured against the activity of the wild type promoter under similar conditions. Promoter activity is generally measured as a function of gene expression and may be determined in a variety of ways including gene expression profiling, measurement of the level of protein expression by SDS gel or other means, or the measurement of reporter activity where reporter gene fusions have been created.

Isolation of Homologs of YAT1 and the YAT1 Putative Promoter Region

It will be appreciated by a person of skill in the art that the promoter regions of the present invention have homologs in a variety of yeast species; and, the use of the promoters for regulated, heterologous gene expression are not limited to those promoters derived from Yarrowia lipolytica, but extend to homologs in other yeast species. For example, the invention encompasses homologs derived from oleaginous genera including, but not limited to: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces; examples of preferred species within these genera include: Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus and R. graminis.

Homology typically is measured using sequence analysis software, wherein the term “sequence analysis software” refers to any computer algorithm or software program (commercially available or independently developed) that is useful for the analysis of nucleotide or amino acid sequences. In general, such computer software matches similar sequences by assigning degrees of homology to various substitutions, deletions and other modifications.

As is well known in the art, isolation of homologous promoter regions using sequence-dependent protocols is readily possible using various techniques; and, these techniques can rely on either the direct identification of a promoter having homology to the YAT1 promoter of the invention or the indirect identification of a promoter by initial identification of gene having significant homology to the yat1 gene and then analysis of the 5′ upstream sequence of the homologous gene. Examples of sequence-dependent protocols include, but are not limited to: 1.) methods of nucleic acid hybridization; 2.) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies [e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or strand displacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci. U.S.A., 89:392 (1992)]; and 3.) methods of library construction and screening by complementation.

For example, putative promoter regions to those of the instant invention could be isolated by using all or a portion of the YAT1 nucleic acid molecules (e.g., corresponding to the promoter, gene or a combination thereof) as DNA hybridization probes to screen libraries from any desired microbe using methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis, supra). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan (e.g., random primers DNA labeling, nick translation, or end-labeling techniques), or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part of (or full-length of) the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full-length DNA fragments under conditions of appropriate stringency.

Typically, in PCR-type amplification techniques, the primers have different sequences and are not complementary to each other. Depending on the desired test conditions, the sequences of the primers should be designed to provide for both efficient and faithful replication of the target nucleic acid. Methods of PCR primer design are common and well known in the art (Thein and Wallace, “The use of oligonucleotides as specific hybridization probes in the Diagnosis of Genetic Disorders”, in Human Genetic Diseases: A Practical Approach, K. E. Davis (Ed.), (1986) pp 33-50 IRL: Herndon, Va.; and Rychlik, W., In Methods in Molecular Biology, White, B. A. (Ed.), (1993) Vol. 15, pp 31-39, PCR Protocols: Current Methods and Applications. Humania: Totowa, N.J.).

Generally two short segments of the instant sequences may be used in PCR protocols to amplify longer nucleic acid molecules encoding homologous polynucleotides from DNA or RNA. The PCR may also be performed on a library of cloned nucleic acid molecules wherein the sequence of one primer is derived from the instant nucleic acid molecules, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the mRNA precursor encoding microbial genes.

Alternatively, the instant sequences may be employed as hybridization reagents for the identification of homologs. The basic components of a nucleic acid hybridization test include a probe, a sample suspected of containing the nucleotide sequence of interest, and a specific hybridization method. Probes of the present invention are typically single-stranded nucleic acid sequences that are complementary to the nucleic acid sequences to be detected. Probes are “hybridizable” to the nucleic acid sequence to be detected. The probe length can vary from 5 bases to tens of thousands of bases, and will depend upon the specific test to be done. Typically a probe length of about 15 bases to about 30 bases is suitable. Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe and the target sequence need not be perfect. Hybridization does occur between imperfectly complementary molecules with the result that a certain fraction of the bases in the hybridized region are not paired with the proper complementary base.

Hybridization methods are well defined. Typically the probe and sample must be mixed under conditions that will permit nucleic acid hybridization. This involves contacting the probe and sample in the presence of an inorganic or organic salt under the proper concentration and temperature conditions. The probe and sample nucleic acids must be in contact for a long enough time that any possible hybridization between the probe and sample nucleic acid may occur. The concentration of probe or target in the mixture will determine the time necessary for hybridization to occur. The higher the probe or target concentration, the shorter the hybridization incubation time needed. Optionally, a chaotropic agent may be added. The chaotropic agent stabilizes nucleic acids by inhibiting nuclease activity. Furthermore, the chaotropic agent allows sensitive and stringent hybridization of short oligonucleotide probes at room temperature (Van Ness and Chen, Nucl. Acids Res. 19:5143-5151 (1991)). Suitable chaotropic agents include guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide and cesium trifluoroacetate, among others. Typically, the chaotropic agent will be present at a final concentration of about 3 M. If desired, one can add formamide to the hybridization mixture, typically 30-50% (v/v).

Various hybridization solutions can be employed. Typically, these comprise from about 20 to 60% volume, preferably 30%, of a polar organic solvent. A common hybridization solution employs about 30-50% v/v formamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 M buffers (e.g., sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), or between 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal), polyvinylpyrrolidone (about 250-500 kdal) and serum albumin. Also included in the typical hybridization solution will be unlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or salmon sperm DNA, or yeast RNA), and optionally from about 0.5 to 2% wt/vol glycine. Other additives may also be included, such as volume exclusion agents that include a variety of polar water-soluble or swellable agents (e.g., polyethylene glycol), anionic polymers (e.g., polyacrylate or polymethylacrylate) and anionic saccharidic polymers (e.g., dextran sulfate).

Recombinant Expression in Yeast

Initiation control regions or promoter regions that are useful to drive expression of a coding gene of interest in the desired host cell are selected from those derived from the upstream portion of the yat1 gene (SEQ ID NO:1). The promoter regions may be identified from the upstream sequences of yat1 and its homologs and isolated according to common methods (Maniatis, supra). Once a promoter region is identified and isolated (e.g., SEQ ID NO:3), it may be operably linked to a coding region of interest to be expressed in a suitable expression vector. These chimeric genes may then be expressed in natural host cells and heterologous host cells, particularly in the cells of oleaginous yeast hosts. Thus, one aspect of the present invention provides a recombinant expression vector comprising a yeast promoter of the invention.

In a further aspect, the invention provides a method of expressing a coding region of interest in a transformed yeast, wherein a transformed yeast is provided having a chimeric gene comprising: (i) a promoter region of a Yarrowia yat1 gene; and, (ii) a coding region of interest expressible in the yeast, wherein the promoter region is operably linked to the coding region of interest; and the transformed yeast is grown under conditions wherein the chimeric gene is expressed. The polypeptide so produced can optionally be recovered from the culture.

Microbial expression systems and expression vectors are well known to those skilled in the art. Any of these could be used to construct chimeric genes comprising a promoter region derived from the yat1 gene for production of any specific coding region of interest suitable for regulated expression in a desirable yeast host cell. These chimeric genes could then be introduced into appropriate microorganisms by integration via transformation to provide expression of the enzymes upon induction. Alternatively, the promoters can be cloned into a plasmid that is capable of transforming and replicating itself in the preferred yeast. The coding region of interest to be expressed can then be cloned downstream from the promoter. Once the recombinant host is established, regulated gene expression can be accomplished by growing the cells under suitable conditions (infra).

Suitable Coding Regions of Interest

Useful chimeric genes will include the promoter region of the yat1 gene as defined herein or a mutant promoter thereof, operably linked to a suitable coding region of interest to be expressed in a preferred host cell.

Coding regions of interest to be expressed in the recombinant yeast host may be either endogenous to the host or heterologous and must be compatible with the host organism. Genes encoding proteins of commercial value are particularly suitable for expression. For example, suitable coding regions of interest may include (but are not limited to) those encoding viral, bacterial, fungal, plant, insect, or vertebrate coding regions of interest, including mammalian polypeptides. Further, these coding regions of interest may be, for example, structural proteins, enzymes (e.g., oxidoreductases, transferases, hydrolyases, lyases, isomerases, ligases), or peptides. A non-limiting list includes genes encoding enzymes such as acyltransferases, aminopeptidases, amylases, carbohydrases, carboxypeptidases, catalyases, cellulases, chitinases, cutinases, cyclodextrin glycosyltransferases, deoxyribonucleases, esterases, α-galactosidases, β-glucanases, β-galactosidases, glucoamylases, α-glucosidases, β-glucosidases, invertases, laccases, lipases, mannosidases, mutanases, oxidases, pectinolytic enzymes, peroxidases, phospholipases, phytases, polyphenoloxidases, proteolytic enzymes, ribonucleases, transglutaminases or xylanases.

Preferred in the present invention in some embodiments are coding regions of the enzymes involved in the production of microbial oils, including ω-6 and ω-3 fatty acids. These coding regions include desaturases and elongases (e.g., see WO 2004/101757 for a partial review of available genes in GenBank and/or the patent literature and considerations for choosing a specific polypeptide having desaturase or elongase activity).

Components of Vectors/DNA Cassettes

Vectors or DNA cassettes useful for the transformation of suitable host cells are well known in the art. The specific choice of sequences present in the construct is dependent upon the desired expression products (supra), the nature of the host cell, and the proposed means of separating transformed cells versus non-transformed cells. Typically, however, the vector or cassette contains sequences directing transcription and translation of the relevant gene(s), a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5′ of the gene that controls transcriptional initiation and a region 3′ of the DNA fragment that controls transcriptional termination. It is most preferred when both control regions are derived from genes from the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host.

Nucleotide sequences surrounding the translational initiation codon ‘ATG’ have been found to affect expression in yeast cells. If the desired polypeptide is poorly expressed in yeast, the nucleotide sequences of exogenous genes can be modified to include an efficient yeast translation initiation sequence motif to obtain optimal gene expression. For expression in yeast, this can be done by site-directed mutagenesis of an inefficiently expressed gene to include the favored translation initiation motif.

The termination region can be derived from the 3′ region of the gene from which the initiation region was obtained or from a different gene. A large number of termination regions are known and function satisfactorily in a variety of hosts (when utilized both in the same and different genera and species from where they were derived). The termination region usually is selected more as a matter of convenience rather than because of any particular property. Preferably, the termination region is derived from a yeast gene, particularly Saccharomyces, Schizosaccharomyces, Candida, Yarrowia or Kluyveromyces. The 3′-regions of mammalian genes encoding γ-interferon and α-2 interferon are also known to function in yeast. Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary; however, it is most preferred if included.

As one of skill in the art is aware, merely inserting a chimeric gene into a cloning vector does not ensure that it will be successfully expressed at the level needed. In response to needs for high expression rates, many specialized expression vectors have been created by manipulating a number of different genetic elements that control aspects of transcription, translation, protein stability, oxygen limitation and secretion from the host cell. More specifically, some of the molecular features that have been manipulated to control gene expression include: 1.) the nature of the relevant transcriptional promoter and terminator sequences; 2.) whether the gene is plasmid-borne or integrated into the genome of the host cell and the number of copies of the cloned gene [e.g., additional copies a particular coding region of interest (operably linked to the promoter of the instant invention) may be introduced into the host to increase expression]; 3.) the final cellular location of the synthesized foreign protein; 4.) the efficiency of translation in the host organism; 5.) the intrinsic stability of the cloned gene protein within the host cell [e.g., expression of the coding region of interest can be increased by removing/deleting destabilizing sequences from either the mRNA or the encoded protein or by adding stabilizing sequences to the mRNA (U.S. Pat. No. 4,910,141)]; and 6.) the codon usage within the cloned gene, such that its frequency approaches the frequency of preferred codon usage of the host cell [e.g. translational efficiency of the encoded mRNAs can be increased by replacement of codons in the native gene with those for optimal gene expression in the selected host microorganism, to thereby substantially enhance the expression of the foreign gene encoding the polypeptide]. Each of these types of modifications are encompassed in the present invention, as means to further optimize expression of a chimeric gene comprising a promoter region of the yat1 gene as defined herein or a mutant promoter thereof, operably linked to a suitable coding region of interest.

Transformation of Yeast Cells

Once an appropriate chimeric gene has been constructed that is suitable for regulated expression in a yeast cell, it is placed in a plasmid vector capable of autonomous replication in a host cell or it is directly integrated into the genome of the host cell. Integration of expression cassettes can occur randomly within the host genome or can be targeted through the use of constructs containing regions of homology with the host genome sufficient to target recombination with the host locus. Where constructs are targeted to an endogenous locus, all or some of the transcriptional and translational regulatory regions can be provided by the endogenous locus.

Where two or more genes are expressed from separate replicating vectors, it is desirable that each vector has a different means of selection and should lack homology to the other constructs to maintain stable expression and prevent reassortment of elements among constructs. Judicious choice of regulatory regions, selection means and method of propagation of the introduced construct can be experimentally determined so that all introduced genes are expressed at the necessary levels to provide for synthesis of the desired products.

Constructs comprising a coding region of interest may be introduced into a host cell by any standard technique. These techniques include transformation (e.g., lithium acetate transformation [Methods in Enzymology, 194:186-187 (1991)]), protoplast fusion, biolistic impact, electroporation, microinjection, or any other method that introduces the gene of interest into the host cell. More specific teachings applicable for oleaginous yeast (i.e., Yarrowia lipolytica) include U.S. Pat. No. 4,880,741 and No. 5,071,764 and Chen, D. C. et al. (Appl Microbiol Biotechnol. 48(2):232-235 (1997)).

For convenience, a host cell that has been manipulated by any method to take up a DNA sequence (e.g., an expression cassette) will be referred to as “transformed” or “recombinant” herein. The transformed host will have at least one copy of the expression construct and may have two or more, depending upon whether the gene is integrated into the genome, amplified, or is present on an extrachromosomal element having multiple copy numbers. The transformed host cell can be identified by various selection techniques, as described in commonly owned U.S. Ser. No. 10/869,630.

Preferred Hosts

Preferred host cells for expression of coding regions of interest operably linked to the instant promoter fragments herein are yeast cells (where oleaginous yeast are most preferred where the desired use is for the production of microbial oils, infra). Oleaginous yeast are naturally capable of oil synthesis and accumulation, wherein the oil can comprise greater than about 25% of the cellular dry weight, more preferably greater than about 30% of the cellular dry weight, and most preferably greater than about 40% of the cellular dry weight. Genera typically identified as oleaginous yeast include, but are not limited to: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. More specifically, illustrative oil-synthesizing yeast include: Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus, R. graminis and Yarrowia lipolytica (formerly classified as Candida lipolytica).

Most preferred is the oleaginous yeast Yarrowia lipolytica; and, in a further embodiment, most preferred are the Y. lipolytica strains designated as ATCC #20362, ATCC #8862, ATCC #18944, ATCC #76982 and/or LGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol. 82(1):43-9 (2002)). The Y. lipolytica strain designated as ATCC #20362 was the particular strain from which the YAT1 promoter was isolated therefrom.

Industrial Production using Transformed Yeast Expressing a Suitable Coding Region of Interest

In general, media conditions that may be optimized for regulated expression of a particular coding region of interest include the type and amount of carbon source, the type and amount of nitrogen source, the carbon-to-nitrogen ratio, the oxygen level, growth temperature, pH, length of the biomass production phase and the time of cell harvest. Microorganisms of interest, such as oleaginous yeast, are grown in complex media (e.g., yeast extract-peptone-dextrose broth (YPD)) or a defined minimal media that lacks a component necessary for growth and thereby forces selection of the desired expression cassettes (e.g., Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.)).

Fermentation media in the present invention must contain a suitable carbon source. Suitable carbon sources may include, but are not limited to: monosaccharides (e.g., glucose, fructose), disaccharides (e.g., lactose, sucrose), oligosaccharides, polysaccharides (e.g., starch, cellulose or mixtures thereof), sugar alcohols (e.g., glycerol) or mixtures from renewable feedstocks (e.g., cheese whey permeate, cornsteep liquor, sugar beet molasses, barley malt). Additionally, carbon sources may include alkanes, fatty acids, esters of fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids and various commercial sources of fatty acids including vegetable oils (e.g., soybean oil) and animal fats. Additionally, the carbon source may include one-carbon sources (e.g., carbon dioxide, methanol, formaldehyde, formate, carbon-containing amines) for which metabolic conversion into key biochemical intermediates has been demonstrated. Hence it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon-containing sources and will only be limited by the choice of the host organism. Although all of the above mentioned carbon sources and mixtures thereof are expected to be suitable in the present invention, preferred carbon sources are sugars and/or fatty acids. Most preferred is glucose and/or fatty acids containing between 10-22 carbons.

Nitrogen may be supplied from an inorganic (e.g., (NH₄)₂SO₄) or organic source (e.g., urea or glutamate). Although the YAT1 promoter is active in media containing nitrogen (e.g., up to about 0.5% ammonium sulfate), the activity of the promoter increases when the host cell is grown in nitrogen-limiting conditions (e.g., in medium containing very low levels of ammonium, or lacking ammonium). Thus, a preferred medium would be one that contains less than about 0.1% ammonium sulfate, or other suitable ammonium salts.

In a more preferred embodiment, the YAT1 promoter is induced when the host cell is grown in media with a high carbon to nitrogen (i.e., C:N) ratio, such as a high glucose medium containing about 8-12% glucose, and about 0.1% or less ammonium sulfate. These conditions are also sufficient to induce oleaginy in those yeast that are oleaginous (e.g., Yarrowia lipolytica).

In addition to appropriate carbon and nitrogen sources, the fermentation media must also contain suitable minerals, salts, cofactors, buffers, vitamins, and other components known to those skilled in the art suitable for the growth of the microorganism.

Preferred growth media in the present invention are common commercially prepared media, such as minimal media made with Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.). Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science. A suitable pH range for the fermentation is typically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.0 is preferred as the range for the initial growth conditions. The fermentation may be conducted under aerobic or anaerobic conditions, wherein microaerobic conditions are preferred.

Host cells comprising a suitable coding region of interest operably linked to the promoters of the present invention may be cultured using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, small-scale or large-scale fermentation in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing regulated expression of the coding region of interest. Furthermore, where commercial production of a product that relies on the instant genetic chimera is desired, a variety of culture methodologies may be applied. For example, large-scale production of a specific gene product over-expressed from a recombinant host may be produced by a batch, fed-batch or continuous fermentation process, as is well known in the art (see, e.g., Thomas D. Brock in Biotechnoloqy: A Textbook of Industrial Microbiology, 2^(nd) ed., (1989) Sinauer Associates: Sunderland, Mass.; or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227 (1992), each herein incorporated by reference).

DESCRIPTION OF PREFERRED EMBODIMENTS

Although the promoters of the present invention will be suitable for regulated expression of any suitable coding region of interest in an oleaginous yeast, in a preferred embodiment the promoters will be utilized in the development of an oleaginous yeast that accumulates high levels of oils enriched in PUFAs. Toward this end, it is necessary to introduce and express e.g., desaturases and elongases that allow for the synthesis and accumulation of ω-3 and/or ω-6 fatty acids.

The term “fatty acids” refers to long chain aliphatic acids (alkanoic acids) of varying chain lengths, from about C₁₂ to C₂₂ (although both longer and shorter chain-length acids are known). The predominant chain lengths are between C₁₆ and C₂₂. The structure of a fatty acid is represented by a simple notation system of “X:Y”, where X is the total number of carbon (C) atoms and Y is the number of double bonds. Additional details concerning the differentiation between “saturated fatty acids” versus “unsaturated fatty acids”, “monounsaturated fatty acids” versus “polyunsaturated fatty acids” (or “PUFAs”), and “ω-6 fatty acids” (ω-6 or n-6) versus “ω-3 fatty acids” (ω-3 or n-3) are provided in commonly owned U.S. Ser. No. 10/840,579.

Nomenclature used to describe PUFAs in the present disclosure is shown below in Table 2. In the column titled “Shorthand Notation”, the omega-reference system is used to indicate the number of carbons, the number of double bonds and the position of the double bond closest to the omega carbon, counting from the omega carbon (which is numbered 1 for this purpose). The remainder of the Table summarizes the common names of ω-3 and c)-6 fatty acids, the abbreviations that will be used throughout the remainder of the specification, and each compounds' chemical name. TABLE 2 Nomenclature Of Polyunsaturated Fatty Acids Shorthand Common Name Abbreviation Chemical Name Notation Linoleic LA cis-9,12-octadecadienoic 18:2 ω-6 γ-Linoleic GLA cis-6,9,12- 18:3 ω-6 octadecatrienoic Eicosadienoic EDA cis-11,14-eicosadienoic 20:2 ω-6 Dihomo-γ- DGLA cis-8,11,14-eicosatrienoic 20:3 ω-6 Linoleic Arachidonic ARA cis-5,8,11,14- 20:4 ω-6 eicosatetraenoic α-Linolenic ALA cis-9,12,15- 18:3 ω-3 octadecatrienoic Stearidonic STA cis-6,9,12,15- 18:4 ω-3 octadecatetraenoic Eicosatrienoic ETrA cis-11,14,17- 20:3 ω-3 eicosatrienoic Eicosatetraenoic ETA cis-8,11,14,17- 20:4 ω-3 eicosatetraenoic Eicosapentaenoic EPA cis-5,8,11,14,17- 20:5 ω-3 eicosapentaenoic Docosapentaenoic DPA cis-7,10,13,16,19- 22:5 ω-3 docosapentaenoic Docosahexaenoic DHA cis-4,7,10,13,16,19- 22:6 ω-3 docosahexaenoic Microbial Biosynthesis of Omega-3 and Omega-6 Fatty Acids

The process of de novo synthesis of palmitate (16:0) in oleaginous microorganisms is described in commonly owned U.S. Ser. No. 10/840,579. This fatty acid is the precursor of longer-chain saturated and unsaturated fatty acid derivates, which are formed through the action of elongases and desaturases. For example, palmitate is converted to its unsaturated derivative [palmitoleic acid (16:1)] by the action of a Δ9 desaturase; similarly, palmitate is elongated to form stearic acid (18:0), which can be converted to its unsaturated derivative by a Δ9 desaturase to thereby yield oleic (18:1) acid.

The metabolic process that converts LA to GLA, DGLA and ARA (the ω-6 pathway) and ALA to STA, ETA, EPA and DHA (the ω-3 pathway) is well described in the literature and is schematically depicted in FIG. 5 (see also commonly owned U.S. Ser. No. 10/840,579 and U.S. Ser. No. 10/869,630). Simplistically, this process involves elongation of the carbon chain through the addition of carbon atoms and desaturation of the molecule through the addition of double bonds, via a series of special desaturation and elongation enzymes present in the endoplasmic reticulim membrane (and hereinafter referred to as “PUFA biosynthetic pathway enzymes”).

More specifically, “PUFA biosynthetic pathway enzymes” or “ω-3/ω-6 biosynthetic pathway enzymes” will refer to any of the following enzymes (and genes which encode said enzymes) associated with the biosynthesis of a PUFA, including: a Δ4 desaturase, a Δ5 desaturase, a Δ6 desaturase, a Δ12 desaturase, a Δ15 desaturase, a Δ17 desaturase, a Δ9 desaturase, a Δ8 desaturase and/or an elongase(s). For further clarity within the present disclosure, the term “desaturase” refers to a polypeptide that can desaturate one or more fatty acids to produce a mono- or polyunsaturated fatty acid or precursor of interest. Thus, despite use of the omega-reference system to refer to specific fatty acids, it is more convenient to indicate the activity of a desaturase by counting from the carboxyl end of the source using the delta-system. For example, a Δ17 desaturase will desaturate a fatty acid between the 17^(th) and 18^(th) carbon atom numbered from the carboxyl-terminal end of the molecule and can, for example, catalyze the conversion of ARA to EPA and/or DGLA to ETA. In contrast, the term “elongase” refers to a polypeptide that can elongate a fatty acid carbon chain to produce a mono- or polyunsaturated fatty acid that is 2 carbons longer than the fatty acid source that the elongase acts upon. This process of elongation occurs in a multi-step mechanism in association with fatty acid synthase, whereby CoA is the acyl carrier (Lassner et al., The Plant Cell 8:281-292 (1996)).

As will be understood by one skilled in the art, the particular functionalities required to be introduced into a host organism for production of a particular PUFA final product will depend on the host cell (and its native PUFA profile and/or desaturase/elongase profile), the availability of substrate and the desired end product(s). As shown in FIG. 5, LA, GLA, EDA, DGLA, ARA, ALA, STA, ETrA, ETA, EPA, DPA and DHA may all be produced in oleaginous yeast, by introducing various combinations of the following PUFA enzyme functionalities: a Δ4 desaturase, a Δ5 desaturase, a Δ6 desaturase, a Δ12 desaturase, a Δ15 desaturase, a Δ17 desaturase, a Δ9 desaturase, a Δ8 desaturase and/or an elongase(s). One skilled in the art will be able to identify various candidate genes encoding each of the above enzymes, according to publicly available literature (e.g., GenBank), the patent literature, and experimental analysis of microorganisms having the ability to produce PUFAs. Thus, a variety of desaturases and elongases are suitable as coding regions of interest in the present invention. These coding regions of interest could be operably linked to the YAT1 promoters of the present invention or mutant promoters thereof, and used as chimeric genes for expression of various ω-6 and ω-3 fatty acids, using techniques well known to those skilled in the art (e.g., see WO 2004/101757 and co-pending U.S. patent application Ser. No. 60/624,812). As such, the invention provides a method for the production of ω-3 and/or ω-6 fatty acids comprising:

-   -   a) providing a transformed oleaginous yeast comprising a genetic         construct, said construct comprising:         -   1) a promoter region of a Yarrowia yat1 gene; and,         -   2) a coding region of interest encoding at least one enzyme             of the ω-3/ω-6 fatty acid biosynthetic pathway;         -   wherein the promoter region and coding region are operably             linked;     -   b) culturing the transformed oleaginous yeast of step (a) under         conditions whereby the at least one enzyme of the ω-3/ω-6 fatty         acid biosynthetic pathway is expressed and a ω-3 or ω-6 fatty         acid is produced; and,     -   c) optionally recovering the ω-3 or ω-6 fatty acid.

In preferred embodiments, the nucleic acid sequence of the promoter region is selected from the group consisting of: SEQ ID NO:3, and subsequences and mutant promoters thereof; the coding region of interest is any desaturase or elongase suitable for regulated expression in the oleaginous yeast for the production of ω-3 or ω-6 fatty acids; and, the transformed oleaginous yeast is cultured under conditions that induce oleaginy.

More specifically, for production of the greatest and the most economical yield of PUFAs, the transformed oleaginous yeast host cell is grown under conditions that optimize desaturase and elongase activities by optimizing expression of the chimeric genes of the present invention, wherein these chimeric genes comprise a promoter region of a yat1 gene and a coding region of interest encoding a PUFA biosynthetic pathway enzyme. Typically, accumulation of high levels of PUFAs in oleaginous yeast cells requires a two-stage process, since the metabolic state must be “balanced” between growth and synthesis/storage of fats. Thus, most preferably, a two-stage fermentation process is necessary for the production of PUFAs in oleaginous yeast. In this approach, the first stage of the fermentation is dedicated to the generation and accumulation of cell mass and is characterized by rapid cell growth and cell division. In the second stage of the fermentation, it is preferable to establish conditions of nitrogen deprivation in the culture to promote high levels of lipid accumulation.

The effect of this nitrogen deprivation is two-fold. First, the nitrogen deprivation reduces the effective concentration of AMP in the cells, thereby reducing the activity of the NAD-dependent isocitrate dehydrogenase of mitochondria. When this occurs, citric acid will accumulate, thus forming abundant pools of acetyl-CoA in the cytoplasm and priming fatty acid synthesis. Secondly, the nitrogen deprivation induces the YAT1 promoter of the invention, thereby promoting expression of any chimeric genes of the invention comprising the YAT1 promoter and a coding region of interest encoding an enzyme of the ω-3/ω-6 fatty acid biosynthetic pathway. Thus, this second phase of the 2-stage fermentation is characterized by the cessation of cell division followed by the synthesis of fatty acids and accumulation of oil.

Although cells are typically grown at about 30° C., some studies have shown increased synthesis of unsaturated fatty acids at lower temperatures (Yongmanitchai and Ward, Appl. Environ. Microbiol. 57:419-25 (1991)). Based on process economics, this temperature shift should likely occur after the first phase of the two-stage fermentation, when the bulk of the organisms' growth has occurred.

Additionally, particular attention is given to several metal ions (e.g., Mn⁺², Co⁺², Zn⁺², Mg⁺²) that promote synthesis of lipids and PUFAs in the fermentation media (Nakahara, T. et al. Ind. Appl. Single Cell Oils, D. J. Kyle and R. Colin, eds. pp 61-97 (1992)).

Purification of PUFAs

The PUFAs produced in a host microorganism as described herein may be found as free fatty acids or in esterified forms such as acylglycerols, phospholipids, sulfolipids or glycolipids, and may be extracted from the host cell through a variety of means well-known in the art. One review of extraction techniques, quality analysis and acceptability standards for yeast lipids is that of Z. Jacobs (Critical Reviews in Biotechnology 12(5/6):463-491 (1992)). A brief review of downstream processing is also available by A. Singh and O. Ward (Adv. Appl. Microbiol. 45:271-312 (1997)).

In general, means for the purification of fatty acids (including PUFAs) may include extraction with organic solvents, sonication, supercritical fluid extraction (e.g., using carbon dioxide), saponification and physical means such as presses, or combinations thereof example of which are contained in commonly owned U.S. Ser. No. 10/840,579.

EXAMPLES

The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

General Methods

Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by: 1.) Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); 2.) T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984); and 3.) Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth of microbial cultures are well known in the art. Techniques suitable for use in the following examples may be found as set out in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, Eds), American Society for Microbiology: Washington, D.C. (1994)); or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2^(nd) ed., Sinauer Associates: Sunderland, Mass. (1989). All reagents, restriction enzymes and materials used for the growth and maintenance of microbial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.) or Sigma Chemical Company (St. Louis, Mo.), unless otherwise specified.

General molecular cloning was performed according to standard methods (Sambrook et al., supra). Oligonucleotides were synthesized by Sigma-Genosys (Spring, Tex.). When polymerase chain reaction (PCR) was involved in subcloning, the constructs were sequenced to confirm that no errors had been introduced to the sequence. PCR products were cloned into Promega's pGEM-T-easy vector (Madison, Wis.).

Manipulations of genetic sequences were accomplished using the suite of programs available from the Genetics Computer Group Inc. (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.). The GCG program “Pileup” was used with the gap creation default value of 12, and the gap extension default value of 4. The GCG “Gap” or “Besffit” programs were used with the default gap creation penalty of 50 and the default gap extension penalty of 3. Unless otherwise stated, in all other cases GCG program default parameters were used.

The meaning of abbreviations is as follows: “sec” means second(s), “min” means minute(s), “h” means hour(s), “d” means day(s), “μL” means microliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” means micromolar, “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “μmole” mean micromole(s), “g” means gram(s), “μg” means microgram(s), “ng” means nanogram(s), “U” means unit(s), “bp” means base pair(s) and “kB” means kilobase(s).

Transformation and Cultivation of Yarrowia Lipolytica

Y. lipolytica strain ATCC #20362 was purchased from the American Type Culture Collection (Rockville, Md.). Strains were usually grown at 28° C. on YPD agar (1% yeast extract, 2% bactopeptone, 2% glucose, 2% agar) or in YPD liquid medium (2% bacto-yeast extract, 3% bactopeptone, 2% glucose).

Transformation of Y. lipolytica was performed according to the method of Chen, D. C. et al. (Appl. Microbiol Biotechnol. 48(2):232-235 (1997)), unless otherwise noted. Briefly, Yarrowia was streaked onto a YPD plate and grown at 30° C. for approximately 18 hr. Several large loopfuls of cells were scraped from the plate and resuspended in 1 mL of transformation buffer containing: 2.25 mL of 50% PEG, average MW 3350; 0.125 mL of 2 M Li acetate, pH 6.0; 0.125 mL of 2 M DTT; and 50 μg sheared salmon sperm DNA. Then, approximately 500 ng of linearized plasmid DNA was incubated in 100 μl of resuspended cells, and maintained at 39° C. for 1 hr with vortex mixing at 15 min intervals. The cells were plated onto selection media plates and maintained at 30° C. for 2 to 3 days.

For selection of transformants, minimal medium (“MM”) was generally used; the composition of MM is as follows: 0.17% yeast nitrogen base (DIFCO Laboratories, Detroit, Mich.) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, pH 6.1 and 20 g/L agar). Supplements of uracil or leucine were added as appropriate to a final concentration of 0.01% (thereby producing “MMU” selection media or “MMLe” selection media).

Alternatively, transformants were selected on 5-fluoroorotic acid (“FOA”; also 5-fluorouracil-6-carboxylic acid monohydrate) selection media, comprising: 0.17% yeast nitrogen base (DIFCO Laboratories) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, 75 mg/L uracil, 75 mg/L uridine, 900 mg/L FOA (Zymo Research Corp., Orange, Calif.) and 20 g/L agar.

“SD” media comprises: 0.67% yeast nitrogen base with ammonium sulfate, without amino acids and 2% glucose. And finally, to promote conditions of oleaginy, High Glucose Media (“HGM”) was prepared as follows: 14 g/L KH₂PO₄, 4 g/L K₂HPO₄, 2 g/L MgSO₄.7H₂O, 80 g/L glucose (pH 6.5).

Fatty Acid Analysis of Yarrowia Lipolytica

For fatty acid analysis, cells were collected by centrifugation and lipids were extracted as described in Bligh, E. G. & Dyer, W. J. (Can. J. Biochem. Physiol. 37:911-917 (1959)). Fatty acid methyl esters were prepared by transesterification of the lipid extract with sodium methoxide (Roughan, G., and Nishida I. Arch Biochem Biophys. 276(1):3846 (1990)) and subsequently analyzed with a Hewlett-Packard 6890 GC fitted with a 30-m×0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard) column. The oven temperature was from 170° C. (25 min hold) to 185° C. at 3.5° C./min.

For direct base transesterification, Yarrowia culture (3 mL) was harvested, washed once in distilled water, and dried under vacuum in a Speed-Vac for 5-10 min. Sodium methoxide (100 μl of 1%) was added to the sample, and then the sample was vortexed and rocked for 20 min. After adding 3 drops of 1 M NaCl and 400 μl hexane, the sample was vortexed and spun. The upper layer was removed and analyzed by GC as described above.

Example 1 Construction and Sequencing of a Yarrowia Lipolytica cDNA Library

The present Example describes the construction of a cDNA library of Y. lipolytica, grown under oleaginous conditions. More specifically, Y. lipolytica cells accumulate considerable amounts of oil when switched to a medium with a high carbon to nitrogen ratio (>80). A cDNA library was constructed from cells grown in an oleaginous medium containing no nitrogen source for 4 hrs, using the BD-Clontech Creator Smart® cDNA library kit (Mississauga, ON, Canada) according to the manufacturer's protocol.

Synthesis of the cDNA library first required growth of Y. lipolytica strain ATCC #20362 in 30 mL YPD medium overnight at 30° C. with shaking. Cells were diluted in two cultures of 100 mL fresh YPD to OD₆₀₀=0.4, then grown until OD₆₀₀=1.5 (Perkin-Elmer Lambda 20 UV/VIS Spectrophotometer). Cells from each culture were pelleted twice by centrifugation at 3750 rpm in a Beckman GH3.8 rotor for 5 min and washed with sterile water, then resuspended in 100 mL HGM. Cells were then shaken at 30° C. for 4 hrs. Each 100 mL culture was divided into three equal portions and pelleted by centrifugation at 3750 rpm in a Beckman GH3.8 rotor for 5 min.

Total RNA was extracted from the pellets using the Qiagen Rneasy Midi Kit. Cells were resusupended in 600 μl buffer RLT from the kit, with β-mercaptoethanol added at a concentration of 10 μl/mL. Resuspended cells were transferred to six 2 mL screw cap tubes each containing 0.6 mL of 0.5 mm glass beads. The cells were homogenized at the HOMOGENIZE-setting on a Biospec (Bartlesville, Okla.) mini bead beater for 2 min. The tubes were briefly spun to settle the beads. Liquid was transferred to 4 fresh 2 mL microfuge tubes and 600 μl of the RLT/BME mix was added to the beads. The bead tubes were vortexed and all liquid above the beads was transferred to the fresh 2 mL tubes. The fresh tubes were spun for 2 min in a microfuge to pellet cells debris, and the supernatent from each set of three tubes that came from a single culture were pooled in a 10 mL screwcap centrifuge tube. The procedure was then completed following the manufacturer's protocol. The two total RNA samples were then combined and mRNA was isolated from the combined sample using the Qiagen Oligotex Midi kit, following the manufacturer's protocol. Purified poly(A)+RNA was obtained with a concentration of 35.7 ng/μl.

cDNA was generated, using the LD-PCR method specified by BD-Clontech and 0.1 μg of polyA(+) RNA sample. Specifically, for 1^(st) strand cDNA synthesis, 3 μl of the poly(A)+RNA sample was mixed with 1 μl of SMART IV oligo nucleotide (SEQ ID NO:4) and 1 μl of CDSIII/3′ PCR primer (SEQ ID NO:5). The mixture was heated at 72° C. for 2 min and cooled on ice for 2 min. To the tube was added the following: 2 μl 1^(st) strand buffer, 1 μl 20 mM DTT, 1 μl 10 mM dNTP mix and 1 μl Powerscript reverse transcriptase. The mixture was incubated at 42° C. for 1 hr and cooled on ice.

The 1^(st) strand cDNA synthesis mixture was used as template for the PCR reaction. Specifically, the reaction mixture contained the following: 2 μl of the 1^(st) strand cDNA mixture, 2 μl 5′-PCR primer (SEQ ID NO:6), 2 μl CDSIII/3′-PCR primer (SEQ ID NO:5), 80 μl water, 10 μl 10× Advantage 2 PCR buffer, 2 μl 50×dNTP mix and 2 μl 50× Advantage 2 polymerase mix. The thermocycler conditions were set for 95° C. for 20 sec, followed by 14 cycles of 95° C. for 5 sec and 68° C. for 6 min on a GenAmp 9600 instrument. PCR product was quantitated by agarose gel electrophoresis and ethidium bromide staining.

Seventy-five μl of the above PCR products (cDNA) were mixed with 3 μl of 20 μg/μl proteinase K supplied with the kit. The mixture was incubated at 45° C. for 20 min, then 75 μl of water was added and the mixture was extracted with 150 μl phenol:chloroform:isoamyl alcohol mixture (25:24:1). The aqueous phase was further extracted with 150 μl chloroform:isoamyl alcohol (25:1). The aqueous phase was then mixed with 15 μl of 3 M sodium acetate, 2 μl of 20 μg/μl glycogen and 400 μl of 100% ethanol. The mixture was immediately centrifuged at room temperature for 20 min at 14000 rpm in a microfuge. The pellet was washed once with 150 μl of 80% ethanol, air dried and dissolved in 79 μl of water.

Dissolved cDNA was subsequently digested with Sfil (79 μl of the cDNA was mixed with 10 μl of 10× Sfil buffer, 10 μl of Sfil enzyme and 1 μl of 100×BSA and the mixture was incubated at 50° C. for 2 hrs). Xylene cyanol dye (2 μl of 1%) was added. The mixture was then fractionated on the Chroma Spin-400 column provided with the kit, following the manufacturer's procedure exactly. Fractions collected from the column were analyzed by agarose gel electrophoresis. The first three fractions containing cDNA were pooled and cDNA precipitated with ethanol. The precipitated cDNA was redissolved in 7 μl of water, and ligated into kit-supplied pDNR-LIB.

Library Sequencing

The ligation products were used to transform E. coli XL-1 Blue electroporation competent cells (Stratagene). An estimated total of 5.4×10⁶ colonies was obtained. Sequencing of the cDNA library was carried out by Agencourt Bioscience Corporation (Beverly, Mass.), using a M13 forward primer (SEQ ID NO:7)

Example 2 Identification of the Yarrowia lipolytica Gene Encoding YAT1 as a Highly Expressed Gene Under Oleaginous Conditions

This example describes the identification of YAT1 as a highly expressed gene under oleaginous conditions. Specifically, the relative abundance of each species of cDNA was examined (based on the sequencing results from Example 1) to identify those genes that were highly expressed under oleaginous conditions. One of the genes that appeared multiple times (16/9984 or 0.16%) was Yali0E27203 (GenBank Accession No. XM_(—)504457; SEQ ID NO:1 herein), annotated as a homolog of the high affinity, low capacity ammonia transporter MEP2 of Saccharomyces cerevisiae (GenBank Accession No. X83608; see also A. M. Marini et al., EMBO J., 13(15):3456-3463 (1994); A. M. Marini et al., Mol Cell Biol 17(8):4282-93 (1997)).

BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1993)) searches were conducted using the corresponding amino acid sequence of Yali0E27203 (SEQ ID NO:2) as a query against the S. cerevisiae MEP1, MEP2 and MEP3-isozymes. The results of these BLAST comparisons are shown below in Table 3 and are reported according to the % identity, % similarity and Expectation value. TABLE 3 Comparison Of Yarrowia lipolytica Homolog To Saccharomyces cerevisiae MEP1, MEP2 And MEP3 % % Similarity Identified Identity^(a) Similarity^(b) E-value^(c) MEP1 35 56 1.8e−79 [GenBank Accession No. X77608] MEP2 52 66 1.9e−132 [GenBank Accession No. X83608] MEP3 37 57 3.6e−83 [GenBank Accession No. P53390] ^(a)% Identity is defined as percentage of amino acids that are identical between the two proteins. ^(b)% Similarity is defined as percentage of amino acids that are identical or conserved between the two proteins. ^(c)Expect value. The Expect value estimates the statistical significance of the match, specifying the number of matches, with a given score, that are expected in a search of a database of this size absolutely by chance. It was hypothesized that YALI0E27203 encoded a Y. lipolytica ammonia transporter of high affinity; thus, YALI0E27203 was tentatively named yat1.

Example 3 Isolation of the 5′ Upstream Region of YAT1 from Yarrowia Lipolytica

To isolate the YAT1 promoter region upstream of the yat1 gene identified in Example 2, primers 27203-F and 27203-R (SEQ ID NOs:8 and 9) were designed, based on the sequence of Y. lipolytica chromosome E between positions 3,222,879 and 3,223,875. These primers were expected to amplify 778 bp of the 5′ upstream untranslated region in front of the ‘ATG’ translation initiation codon of the yat1 gene, from −778 to −1 (wherein the ‘A’ position of the ‘ATG’ translation initiation codon was designated as +1).

PCR amplification was performed in a 50 μl total volume using a 1:1 dilution of a premixed 2×PCR solution (TaKaRa ExTaq, TaKaRa Bio Inc., Otsu Shiga, 520-2193, Japan). The final composition contained 25 mM TAPS, pH 9.3, 50 mM KCl, 2 mM MgCl₂, 1 mM 2-mercaptoethanol, 200 μM each deoxyribonucleotide triphosphate, 10 pmole each primer (supra), 50 ng Y. lipolytica ATCC #20362 genomic DNA, and 1.25 units of TaKaRa ExTaq™ DNA polymerase. The reaction mixture was first heated to 94° C. for 150 sec. Amplification was carried out for 30 cycles at 94° C. for 30 sec, 55° C. for 30 sec and 72° C. for 1 min. This was followed by a final extension for 7 min at 72° C.

The PCR product was analyzed by agarose gel electrophoresis and was shown to contain a single DNA fragment of ˜800 bp. This fragment was purified using a Qiagen PCR purification kit following the manufacturer's protocol.

Example 4 Synthesis of Plasmids pY5-30, pYGPAT-GUS and pDMW214

A series of plasmids were created comprising a chimeric gene composed of various native Y. lipolytica promoters and the “GUS” reporter gene, wherein “GUS” corresponds to the E. coli gene encoding β-glucuronidase (Jefferson, R. A. Nature. 342(6251):837-838 (1989)). This was required for comparative studies investigating the promoter activity of TEF, YAT1, FBAIN and GPAT, as described in Example 8.

Synthesis of Plasmid pY5-30 (TEF::GUS::XPR)

The synthesis of plasmid pY5-30, comprising a TEF::GUS::XPR chimeric gene, is described in WO2005/003310. More specifically, plasmid pY5-30 (FIG. 1A; SEQ ID NO:10) contained: a Yarrowia autonomous replication sequence (ARS18); a ColE1 plasmid origin of replication; an ampicillin-resistance gene (Amp^(R)) for selection in E. coli; a Yarrowia LEU2 gene for selection in Yarrowia; and the chimeric TEF::GUS::XPR gene.

Synthesis of Plasmid pDMW214 (FBAIN::GUS::XPR)

The synthesis of plasmid pDMW214, comprising a FBAIN::GUS::XPR chimeric gene, is described in WO 2005/049805. Briefly, however, the FBAIN promoter region (SEQ ID NO:11; which includes both an upstream DNA sequence and a downstream sequence from the putative ‘ATG’ translation initiation codon of the fructose-bisphosphate aldolase (fba1) gene [wherein the downstream region comprises an intron]) was amplified by PCR, digested with NcoI and SaII, and then purified following gel electrophoresis. The NcoI/SaIII-digested PCR products were ligated to NcoI/SaII digested pY5-30 vector to produce plasmid “pDMW214” (FIG. 1B).

Synthesis of pYGPAT-GUS (GPAT::GUS::XPR)

The synthesis of plasmid pYGPAT-GUS, comprising a GPAT::GUS::XPR chimeric gene, is described in U.S. patent application Ser. No. 60/610,060 (filed Sep. 15, 2004) and will be reiterated below. In general, synthesis of the plasmid required identification and isolation of the Y. lipolytica glycerol-3-phosphate O-acyltransferase (gpat) gene, isolation of the promoter region upstream of the putative ‘ATG’ translation initiation codon via genome-walking, and then cloning of the promoter into a derivative of pY5-30 (supra).

Isolation and Sequencing of the Putative Yarrowia Lipolytica GPAT

Based on the gene sequences encoding two isozymes of GPAT in Saccharomyces cerevisiae (GAT1 [GenBank Accession No. AJ311354] and GAT2(SCT1) [GenBank Accession No. AJ314608]; see also Zheng and Zou. J. Biol. Chem. 276(45):4171041716 (2001) and WO 02/08391 A2), BLAST searches (supra, Example 2) were conducted against the Genolevures genome database of Y. lipolytica ORFs (sponsored by the Center for Bioinformatics, LaBRI, bâtiment A30, Université Bordeaux 1, 351, cours de la Libération, 33405 Talence Cedex, France) to identify any similar sequences contained therein. The results of the BLAST comparisons identified one homolog (Yarrowia lipolytica ORF YAL1-CDS1055.1, a protein of 727 amino acids; SEQ ID NO:12; also GenBank Accession No. CAG81570) annotated as a protein having similarity to S. cerevisiae GPATs.

Following the tentative identification of ORF YAL1-CDS1055.1, this amino acid sequence was BLASTed against the S. cerevisiae isozymes encoding GPAT. The results of these BLAST comparisons are shown below and are reported according to the % identity, % similarity, and Expectation value (supra, Example 2). TABLE 4 Comparison Of Yarrowia lipolytica ORF YAL1-CDS1055.1 To Saccharomyces cerevisiae GAT1 And GAT2(SCT1) % % Similarity Identified Identity^(a) Similarity^(b) E-value^(c) GAT1 35 54 1.2e−113 [GenBank Accession No. AJ311354] GAT2(SCT1) 43 59 3.4e−141 [GenBank Accession No. AJ314608] Thus, it was hypothesized that ORF YALI-CDS1055.1 encoded a Y. lipolytica GPAT.

Degenerated oligonucleotides, as shown below, were designed to amplify the entire coding region of ORF YALI-CDS1055.1. Degenerated Oligonucleotide YGPAT-F (SEQ ID NO:13) ATGTCNGAGACYGACCAYCTNCTN Degenerated oligonucleotide YGPAT-R (SEQ ID NO:14) YTCYTCRTCYTGYTCTCGYCGYTT [Note: The nucleic acid degeneracy code used for SEQ ID NOs:13 and 14 was as follows: R = A/G; Y = C/T; and N = A/C/T/G.]

The PCR amplification was carried out in a 50 μl total volume using a 1:1 dilution of a premixed 2×PCR solution and 1.25 units of TaKaRa Ex Taq™ DNA polymerase (Takara Mirus Bio, Madison, Wis.), according to the PCR composition described in Example 3. The thermocycler conditions were set for 30 cycles at 94° C. for 2.5 min, 55° C. for 30 sec and 72° C. for 2.5 min, followed by a final extension at 72° C. for 6 min.

The PCR products were separated by gel electrophoresis in 1% (w/v) agarose. A 2.2 kB DNA fragment was excised and purified using a QiaexII gel purification kit (Qiagen, Valencia, Calif.). Subequently, the purified 2.2 kB DNA fragment was cloned into the pGEM-T-easy vector (Promega, Madison, Wis.). The ligated DNA was used to transform cells of E. coli Top10 and transformants were selected on LB (1% bacto-tryptone, 0.5% bacto-yeast extract and 1% NaCl) agar containing ampicillin (100 μg/mL). Analysis of the plasmid DNA from one transformant confirmed the presence of a plasmid of the expected size, designated as “pGPAT-1”.

Sequence analyses of pGPAT-1 showed that it contained a 2184 bp fragment of Y. lipolytica DNA encoding GPAT (SEQ ID NO:15). Specifically, identity of this gene sequence was determined by conducting BLAST searches for similarity to sequences contained in the BLAST “nr” database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the SWISS-PROT protein sequence database, EMBL and DDBJ databases). The sequence was analyzed for similarity to all publicly available DNA sequences contained in the “nr” database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequence was translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the “nr” database, using the BLASTX algorithm (Gish, W. and States, D. J. Nature Genetics 3:266-272 (1993)) provided by the NCBI. The BLAST searches revealed that the translated product of SEQ ID NO:15 (comprising the putative gpat gene) had the highest BLAST hits to annotated GPATs from: (1) S. cerevisiae (SwissPro P36148, Entrez CAA82146): 43% identity, 59% similarity, E-146; and (2) Schizosaccharomyces pombe SCT1(GAT2) homolog (GPAT): 48% identity, 66% similarity, E-148.

Furthermore, the translated product of SEQ ID NO:15 was 100% identical to the amino acid sequence of ORF YAL1-CDS1055.1 (SEQ ID NO:12; also GenBank Accession No. CAG81570).

Isolation of the 5′ Upstream Region of GPAT from Y. Lipolytica

To isolate the GPAT promoter region upstream of the gene identified in Example 1, a genome-walking technique (Universal GenomeWalker, ClonTech, CA) was utilized, following the manufacturer's protocol.

Briefly, genomic DNA of Y. lipolytica was digested with DraI, EcoRV, PvuII or StuI individually, and the digested DNA samples were ligated with Genome Walker adaptor (SEQ ID NOs:16 [top strand] and 17 [bottom strand]), as shown below: 5′-GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT-3′                                     3′-H2N-CCCGACCA-5′

PCR reactions were then carried out using the ligation products as templates, and the following oligonucleotides as primers: Adaptor-1 and YGPAT-5R-1 (SEQ ID NOs:18 and 19). The PCR amplification was carried out in a 50 μl total volume using the components and conditions previously described, with the exception that the template used was 1 μl of ligation product (versus 50 ng genomic DNA). Second PCR reactions were then carried out using 1 μl of 1:50 diluted first PCR product as template, and Nested Adaptor Primer 2 and YGPAT-5R-2 (SEQ ID NOs:20 and 21) as primers.

A 1.7 kB DNA fragment, amplified from the EcoRV digested sample, was purified using a Qiagen PCR purification kit and cloned into the pGEM-T-easy vector (Promega, Madison, Wis.). The ligated DNA was used to transform E. coli Top10 and transformants were selected on LB agar containing ampicillin (100 μg/mL).

Analysis of the plasmid DNA from one transformant confirmed the presence of the expected plasmid, designated “pEcoRV-G-5”. Sequence analyses showed that pEcoRV-G-5 contained a fragment of 1781 bp (SEQ ID NO:22), which included 1678 bp of 5′ upstream sequence from the nucleotide ‘A’ of the translation initiation codon ‘ATG’ of the GPAT gene. Assembly of DNA corresponding to overlapping SEQ ID NOs:15 and 22 yielded a single contig of DNA represented as SEQ ID NO:23 (FIG. 2; 3862 bp total length). This contig therefore contained the −1678 to +2181 region of the GPAT gene, wherein the ‘A’ position of the ‘ATG’ translation initiation codon was designated as +1.

Construction of pYGPAT-GUS

The nucleotide region between the −1130 position and the ‘ATG’ translation initiation site of the gpat gene was considered to represent the putative GPAT promoter region (“GPAT-Pro”, provided as SEQ ID NO:24).

GPAT-Pro was amplified by PCR using primers GPAT-5-1 and GPAT-5-2 (SEQ ID NOs:25 and 26), and pEcoRV-G5 (supra) as template. The PCR product was then purified using a Qiagen PCR purification kit and was completely digested with SaII and NcoI. The digestion product was again purified with a Qiagen PCR purification kit and ligated to NcoI/SaII digested pY5-30 vector (supra, wherein the NcoI/SaII digestion had excised the TEF promoter from the pY5-30 vector backbone). Ligated DNA was then used to individually transform E. coli Top10. Transformants were selected on LB agar containing ampicillin (100 μg/mL).

Analysis of the plasmid DNA from one transformant containing GPAT-Pro confirmed the presence of the expected plasmid, designated “pYGPAT-GUS”. Thus, this plasmid contained a chimeric gene comprising a GPAT promoter, GUS reporter gene and XPR terminator (FIG. 1C).

Example 5 Synthesis of pYAT-GUS

The present Example describes the synthesis of pYAT-GUS (comprising a YAT1::GUS::XPR chimeric gene). Synthesis of this plasmid first required amplification of the putative YAT1 promoter region. Then, the putative promoter region was cloned into pYGPAT-GUS (supra, Example 4).

The purified YAT1 PCR product from Example 3 was digested with HindIII and SaII and a ˜600 bp fragment was isolated by agarose gel electrophoresis followed by purification with Qiagen MinElute Gel purification kit according to the manufacturer's protocol. Furthermore, the YAT1 PCR product was also digested with NcoI and HindIII, and a ˜200 bp fragment was isolated and purified as above. Finally, plasmid pYGPAT-GUS was digested with SaII and NcoI, and a ˜9.5 kB fragment was isolated and purified. The three DNA fragments were ligated together to create pYAT-GUS (FIG. 1D). In the resulting plasmid, the YAT1 promoter region (SEQ ID NO:3; corresponding to 778 bp of the 5′ upstream region of YALI0E27203 (−778 to −1)) was fused to the GUS gene, followed by the XPR terminator region.

Example 6 Generation of ARA-Producing Yarrowia lipolytica ATCC #20362 Strain Y2034

The present Example describes the construction of strain Y2034, derived from Y. lipolytica ATCC #20362, capable of producing significant concentrations of ARA relative to the total lipids. Comparison of the TEF, YAT1, GPAT and FBAIN promoters was examined in this ARA-producing strain based on analysis of GUS expression, as described in Example 8 (infra).

The development of strain Y2034 herein, producing 10% ARA, required the construction of strain M4 (producing 8% DGLA) (FIG. 3A).

Construction of Strain M4 Producing 8% DGLA

Construct pKUNF12T6E (FIG. 3B; SEQ ID NO:27) was generated to integrate four chimeric genes (comprising a Δ12 desaturase, a Δ6 desaturase and 2 elongases) into the Ura3 loci of wild type Yarrowia strain ATCC #20362, to thereby enable production of DGLA. The pKUNF12T6E plasmid contained the following components: TABLE 5 Description of Plasmid pKUNF12T6E (SEQ ID NO: 27) RE Sites And Nucleotides Within SEQ ID Description Of Fragment And NO: 27 Chimeric Gene Components AscI/BsiWI 784 bp 5′ part of Yarrowia Ura3 gene (GenBank (9420-8629) Accession No. AJ306421) SphI/PacI 516 bp 3′ part of Yarrowia Ura3 gene (GenBank (12128-1) Accession No. AJ306421) SwaI/BsiWI FBAIN::EL1S:Pex20, comprising: (6380-8629) FBAIN: FBAIN promoter (SEQ ID NO: 11) EL1S: codon-optimized elongase 1 gene (SEQ ID NO: 28), derived from Mortierella alpina (GenBank Accession No. AX464731) Pex20: Pex20 terminator sequence from Yarrowia Pex20 gene (GenBank Accession No. AF054613) BglII/SwaI TEF::Δ6S::Lip1, comprising: (4221-6380) TEF: TEF promoter (GenBank Accession No. AF054508) Δ6S: codon-optimized Δ6 desaturase gene (SEQ ID NO: 30), derived from Mortierella alpina (GenBank Accession No. AF465281) Lip1: Lip1 terminator sequence from Yarrowia Lip1 gene (GenBank Accession No. Z50020) PmeI/ClaI FBA::F.Δ12::Lip2, comprising: (4207-1459) FBA: FBA promoter (SEQ ID NO: 32) F.Δ12: Fusarium moniliforme Δ12 desaturase gene (SEQ ID NO: 33) Lip2: Lip2 terminator sequence from Yarrowia Lip2 gene (GenBank Accession No. AJ012632) ClaI/PacI TEF::EL2S::XPR, comprising: (1459-1) TEF: TEF promoter (GenBank Accession No. AF054508) EL2S: codon-optimized elongase gene (SEQ ID NO: 35), derived from Thraustochytrium aureum (U.S. Pat. No. 6,677,145) XPR: XPR terminator sequence of Yarrowia Xpr gene (GenBank Accession No. M17741)

The pKUNF12T6E plasmid was digested with AscI/SphI, and then used for transformation of wild type Y. lipolytica ATCC #20362 according to the General Methods. The transformant cells were plated onto FOA selection media plates and maintained at 30° C. for 2 to 3 days. The FOA resistant colonies were picked and streaked onto MM and MMU selection plates. The colonies that could grow on MMU plates but not on MM plates were selected as Ura-strains. Single colonies of Ura-strains were then inoculated into liquid MMU at 30° C. and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.

GC analyses showed the presence of DGLA in the transformants containing the 4 chimeric genes of pKUNF12T6E (FIG. 3B), but not in the wild type Yarrowia control strain. Most of the selected 32 Ura⁻ strains produced about 6% DGLA of total lipids. There were 2 strains (i.e., strains M4 and 13-8) that produced about 8% DGLA of total lipids.

Construction of Strain Y2034 Producing 10% ARA

Construct pDMW232 (FIG. 3C; SEQ ID NO:37) was generated to integrate two Δ5 chimeric genes into the Leu2 gene of Yarrowia strain M4.

Plasmid pDMW232 contained the following components, as described in Table 6: TABLE 6 Description of Plasmid pDMW232 (SEQ ID NO: 37) RE Sites And Nucleotides Within SEQ ID Description Of Fragment And NO: 37 Chimeric Gene Components AscI/BsiWI 788 bp 5′ part of Yarrowia Leu2 gene (GenBank (5550-4755) Accession No. AF260230) SphI/PacI 703 bp 3′ part of Yarrowia Leu2 gene (GenBank (8258-8967) Accession No. AF260230) SwaI/BsiWI FBAIN::MAΔ5::Pex20, comprising: (2114-4755) FBAIN: FBAIN Promoter (SEQ ID NO: 11) MAΔ5: Mortierella alpina Δ5 desaturase gene (GenBank Accession No. AF067654) Pex20: Pex20 terminator sequence of Yarrowia Pex20 gene (GenBank Accession No. AF054613) SwaI/ClaI TEF::MAΔ5::Lip1, comprising: (2114-17) TEF: TEF promoter (GenBank Accession No. AF054508) MAΔ5: as described for FBAIN::MAΔ5::Pex20 (supra) Lip1: Lip1 terminator sequence of Yarrowia Lip1 gene (GenBank Accession No. Z50020) PmeI/ClaI Yarrowia Ura3 gene (GenBank Accession No. AJ306421) (5550-4755)

Plasmid pDMW232 was digested with AscI/SphI, and then used to transform strain M4 (Example 4) separately according to the General Methods. Following transformation, the cells were plated onto MMLe plates and maintained at 30° C. for 2 to 3 days. The individual colonies grown on MMLe plates were picked and streaked onto MM and MMLe plates. Those colonies that could grow on MMLe plates but not on MM plates were selected as Leu2⁻ strains. Single colonies of Leu2⁻ strains were then inoculated into liquid MMLe media at 30° C. and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.

GC analyses showed the presence of ARA in pDMW232 transformants, but not in the parental M4 strain. Specifically, among the 48 selected Leu2⁻ transformants with pDMW232, there were 34 strains that produced less than 5% ARA, 11 strains that produced 6-8% ARA, and 3 strains that produced about 10% ARA of total lipids in the engineered Yarrowia. One of the strains that produced 10% ARA was named “Y2034” and was the parental strain in Example 7.

Example 7 Transformation of Y. lipolytica with pY5-30, pYAT-GUS, pYGPAT-GUS and pDMW214

The plasmids pY5-30 (Example 4; comprising a TEF::GUS::XPR chimeric gene), pYAT-GUS (Example 5; comprising a YAT1::GUS::XPR chimeric gene), pYGPAT-GUS (Example 4; comprising a GPAT::GUS::XPR chimeric gene) and pDMW214 (Example 4; comprising a FBAIN::GUS::XPR chimeric gene) were transformed separately into Y. lipolytica ATCC #20362 strain Y2034, according to the General Methods. Selection was performed on SD medium comprising 2.5% agar.

Using this technique, transformants were obtained that contained pY5-30, pYAT-GUS, pYGPAT-GUS and pDMW214, respectively.

Example 8 Comparative Analysis of the TEF, YAT1, GPAT and FBAIN Promoter Activities in Yarrowia Lipolytica, as Determined by Histochemical Assay

The activity of the TEF, YAT1, GPAT and FBAIN promoters was determined in Y. lipolytica containing the pY5-30, pYAT-GUS, pYGPAT-GUS and pDMW214 constructs, each of which possessed a GUS reporter gene and an XPR terminator (from Example 7). GUS activity in each expressed construct was measured by histochemical assays (Jefferson, R. A. Plant Mol. Biol. Reporter 5:387-405 (1987)).

Specifically, Y. lipolytica strains containing plasmids pY5-30, pYAT-GUS, pYGPAT-GUS and pDMW214, respectively, were grown from single colonies in 5 mL SD media at 30° C. for 24 hrs to an OD₆₀₀˜8.0. Then, 1 mL of cells were collected by centrifugation. The remaining cultures were centrifuged and washed 2× with HGM, resuspended in 5 mL each of HGM and allowed to grow at 30° C. further. After 24 and 120 hrs, ˜0.25 mL of each culture were centrifuged to collect the cells. Cell samples were resuspended individually in 100 μl of histochemical staining buffer [Staining buffer prepared by dissolving 5 mg of 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) in 50 μl dimethyl formamide, followed by addition of 5 mL 50 mM NaPO₄, pH 7.0.]. Zymolase 20T (5 μl of 1 mg/mL; ICN Biomedicals, Costa Mesa, Calif.) was added to each, and the mixture incubated at 30° C.

The results of histochemical staining showed that the YAT1 promoter in construct pYAT-GUS was active. Comparatively, the YAT1 promoter appeared to be stronger than the TEF promoter (FIG. 4A) but significantly weaker than the FBAIN promoter and GPAT promoter, when cells were grown in SD medium for 24 hrs. More interestingly, however, it appeared that the YAT1 promoter was stronger than the GPAT promoter and comparable with FBAIN promoter in cells grown in HGM for 24 hrs (FIG. 4B). This remained true after 120 hrs in HGM (FIG. 4C). Thus, the YAT1 promoter appeared to be induced in HGM, a medium that promotes oleaginous growth conditions due to nitrogen limitation.

Example 9 Comparative Analysis of the TEF, YAT1, FBAIN and GPAT Promoter Activities in Yarrowia Lipolytica, as Determined by Fluorometric Assay

A variety of methods are available to compare the activity of various promoters, to thereby facilitate determination of each promoter's strength for use in future applications wherein a suite of promoters would be necessary to construct chimeric genes. Thus, although it may be useful to indirectly quantitate promoter activity based on reporter gene expression using histochemical staining (Example 8), quantification of GUS expression using more quantitative means may be desirable. One suitable method to assay GUS activity is by fluorometric determination of the production of 4-methylumbelliferone (4-MU) from the corresponding β-glucuronide (4-MUG; see Jefferson, R. A., Plant Mol. Biol. Reporter 5:387-405 (1987)).

Yarrowia lipolytica strain Y2034 containing plasmids pY5-30, pYAT-GUS, pYGPAT-GUS and pDMW214 constructs, respectively (from Example 7), were grown from single colonies in 10 mL SD medium at 30° C. for 48 hrs to an OD₆₀₀ ˜5.0. Two mL of each culture was collected for GUS activity assays, as described below, while 5 mL of each culture was switched into HGM.

Specifically, cells from the 5 mL aliquot were collected by centrifugation, washed once with 5 mL of HGM and resuspended in HG medium. The cultures in HGM were then grown in a shaking incubator at 30° C. for 24 hrs. Two mL of each HGM culture were collected for GUS activity assay, while the remaining culture was allowed to grow for an additional 96 hrs before collecting an additional 2 mL of each culture for the assay.

Each 2 mL culture sample in SD medium was resuspended in 1 mL of 0.5×cell culture lysis reagent (Promega). Resuspended cells were mixed with 0.6 mL of glass beads (0.5 mm diameter) in a 2.0 mL screw cap tube with a rubber O-ring. The cells were then homogenized in a Biospec mini beadbeater (Bartlesville, Okla.) at the highest setting for 90 sec. The homogenization mixtures were centrifuged for 2 min at 14,000 rpm in an Eppendof centrifuge to remove cell debris and beads. The supernatant was used for GUS assay and protein determination.

For each fluorometric assay, 200 μl of extract was added to 800 μl of GUS assay buffer (2 mM 4-methylumbelliferyl-β-D-glucuronide (“MUG”) in extraction buffer) and placed at 37° C. Aliquots of 100 μl were taken at 0, 30 and 60 min time points and added to 900 μl of stop buffer (1 M Na₂CO₃). Each time point was read using a Fluorimeter (CytoFluorR Series 4000, Framingham, Mass.) set to an excitation wavelength of 360 nm and an emission wavelength of 455 nm. Total protein concentration of each sample was determined using 20 μl of extract and 980 μl of BioRad Bradford reagent (Bradford, M. M. Anal. Biochem. 72:248-254 (1976)). GUS activity is expressed as nmoles of 4-MU per minute per mg of total protein.

As shown in the Table below, the activity of the YAT1 promoter was highly induced after 24 hrs in HGM. TABLE 7 Comparison of TEF, FBAIN, YAT1 And GPAT Promoter Activity Under Various Growth Conditions Culture Promoter Conditions TEF FBAIN YAT1 GPAT  48 hr, SD 0.401 43.333 0.536 5.252  24 hr, HGM 0.942 30.694 19.154 2.969 120 hr HGM 0.466 17.200 13.400 3.050

Based on the data above wherein the activity of the YAT1 promoter was quantitated based on GUS activity of cell extracts, the activity of the YAT1 promoter increased by ˜37 fold when cells were switched from SD medium into HGM and grown for 24 hrs. After 120 hrs in HGM, the activity was reduced somewhat but was still 25× higher than the activity in SD medium. In contrast, the activity of the FBAIN promoter and the GPAT promoter was reduced by 30% and 40%, respectively, when switched from SD medium into HGM for 24 hrs. The activity of the TEF promoter increased by 2.3 fold after 24 hrs in HGM. Thus, the YAT1 promoter is inducible under oleaginous conditions and will thereby function as an effective promoter for the regulated expression of heterologous genes in Yarrowia lipolytica. 

1. A method for the expression of a coding region of interest in a transformed yeast comprising: c) providing a transformed yeast having a genetic construct comprising: (i) a promoter region of a Yarrowia yat1 gene; and, (ii) a coding region of interest expressible in the yeast cell; wherein the promoter region is operably linked to the coding region of interest; and, d) growing the transformed yeast of step (a) under conditions whereby the genetic construct of step (a) is expressed.
 2. A method according to claim 1 wherein the Yarrowia yat1 gene is isolated from Yarrowia lipolytica.
 3. A method according to claim 1 wherein the promoter region of the yat1 gene is contained within a nucleic acid molecule as set forth in SEQ ID NO:3.
 4. A method according to claim 3 wherein the promoter region contains at least one mutation that does not diminish its promoter activity.
 5. A method according to claim 4 wherein the promoter activity is at least about 20% to at least about 400% of the promoter activity of the wildtype promoter activity.
 6. A method according to claim 5 wherein the promoter activity is determined by histochemical assay.
 7. A method according to claim 1 wherein the transformed yeast is an oleaginous yeast.
 8. A method of claim 7, wherein the oleaginous yeast is a member of a genus selected from the group consisting of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.
 9. A method according to claim 8, wherein the oleaginous yeast is selected from the group consisting of: Yarrowia lipolytica ATCC #20362, Yarrowia lipolytica ATCC #8862, Yarrowia lipolytica ATCC #18944, Yarrowia lipolytica ATCC #76982 and Yarrowia lipolytica LGAM S(7)1.
 10. A method according to claim 1 wherein the coding region of interest encodes a polypeptide selected from the group consisting of: desaturases, elongases, acyltransferases, aminopeptidases, amylases, carbohydrases, carboxypeptidases, catalyases, cellulases, chitinases, cutinases, cyclodextrin glycosyltransferases, deoxyribonucleases, esterases, α-galactosidases, β-galactosidases, glucoamylases, α-glucosidases, β-glucanases, β-glucosidases, invertases, laccases, lipases, mannosidases, mutanases, oxidases, pectinolytic enzymes, peroxidases, phospholipases, phytases, polyphenoloxidases, proteolytic enzymes, ribonucleases, transglutaminases and xylanases.
 11. A method according to claim 1 wherein the conditions whereby the genetic construct of step (a) is expressed are those of nitrogen-limitation.
 12. A method according to claim 11 wherein the conditions of nitrogen-limitation are conditions that induce oleaginy in the transformed yeast.
 13. A method for the production of an ω-3 or an ω-6 fatty acid comprising: d) providing a transformed oleaginous yeast comprising a genetic construct, comprising: (i) a promoter region of a Yarrowia yat1 gene; and, (ii) a coding region encoding at least one enzyme of the ω-3/ω-6 fatty acid biosynthetic pathway; wherein the promoter region and coding region are operably linked; e) culturing the transformed oleaginous yeast of step (a) under conditions whereby the at least one enzyme of the ω-3/ω-6 fatty acid biosynthetic pathway is expressed and a ω-3 or ω-6 fatty acid is produced; and, f) optionally recovering the ω-3 or ω-6 fatty acid.
 14. A method according to claim 13 wherein the Yarrowia yat1 gene is isolated from Yarrowia lipolytica.
 15. A method according to claim 13 wherein the promoter region of the yat1 gene is contained within a nucleic acid molecule as set forth in SEQ ID NO:3.
 16. A method according to claim 13 wherein the coding region of interest encodes a polypeptide selected from the group consisting of: desaturases and elongases.
 17. A method according to claim 16 wherein the desaturase is selected from the group consisting of: Δ9 desaturase, Δ12 desaturase, Δ6 desaturase, Δ5 desaturase, Δ17 desaturase, Δ15 desaturase, Δ8 desaturase and Δ4 desaturase.
 18. A method according to claim 13 wherein the oleaginous yeast is a member of a genus selected from the group of consisting of: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.
 19. A method according to claim 18 wherein the oleaginous yeast is Yarrowia lipolytica.
 20. A method according to claim 19, wherein the Yarrowia lipolytica is a strain selected from the group consisting of: Yarrowia lipolytica ATCC #20362, Yarrowia lipolytica ATCC #8862, Yarrowia lipolytica ATCC #18944, Yarrowia lipolytica ATCC #76982 and Yarrowia lipolytica LGAM S(7)1.
 21. A method according to claim 13 wherein the ω-3 or ω-6 fatty acid is selected from the group consisting of: linoleic acid, α-linolenic acid, γ-linolenic acid, stearidonic acid, dihomo-γ-linoleic acid, eicosatetraenoic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid, eicosadienoic acid and eicosatrienoic acid.
 22. A method according to claim 13 wherein the conditions whereby the at least one enzyme of the ω-3/ω-6 fatty acid biosynthetic pathway is expressed are those of nitrogen limitation.
 23. A method according to claim 22 wherein the conditions of nitrogen-limitation are conditions that induce oleaginy in the transformed oleaginous yeast. 